DIRECT CONVERSION OF N-PROTECTED AMINO ALCOHOLS INTO AMINO ALKYL AZIDES AND SYNTHESIS OF TRIAZOLES
Major project report
Submitted for partial fulfillment for the award of degree of
BACHELOR OF ENGINEERING BE
SANKET W K
Guide : Co-Guide :
Dr. Thomas Theodore
Dr. H. S. Lalithamba
Department of Chemistry,
DEPARTMENT OF CHEMICAL ENGINEERING
SIDDAGANGA INSTITUTE OF TECHNOLOGY, Tumakuru-572103, Karnataka, India
(An Autonomous Institution Affiliated to Visvesvaraya Technological University-Belagavi, Approved
by AICTE, Programmes Accredited by NBA, New Delhi)
SIDDAGANGA INSTITUTE OF TECHNOLOGY
(An Autonomous Institution Affiliated to Visvesvaraya Technological University-Belagavi)
Tumakuru-572103, Karnataka, India
DEPARTMENT OF CHEMICAL ENGINEERING
This is to certify that the major project entitled “DIRECT CONVERSION OF N-PROTECTED AMINO ALCOHOLS INTO AMINO ALKYL AZIDEAND SYNTHESIS OF TRIAZOLES “submitted by MEGHANA D 1SI14CH017, MOHAMMED ZIYA 1SI14CH020, SANKET W K 1SI14CH029 of Department of Chemical Engineering, Siddaganga Institute of Technology, Tumakuru to fulfill the academic requirement of Eighth Semester in Chemical Engineering for the academic year 2017-18, is a bonafide record of fully independent and original work carried out by them, under my supervision during the academic year 2017-2018.
Signature of Guide Signature of Co-Guide Signature of HOD Signature of Principal
(Dr.Thomas (Dr.H S Lalithamba ) (Dr.S Murthy Shekhar) (Dr.Shivakumaraiah)
EXAMINERS Signature with date
We wish to give our sincere and heartfelt gratitude to His Holiness Dr. Sree Sree Sivakumara Swamigalu, President, Siddaganga Institute of Technology, Tumakuru for providing us with congenial and his serene ambience that kept us in good spirits.
We are thankful to Dr. M. N. Channabasappa, Director &Dr. Shivakumaraiah, Principal, SIT, Tumakuru who protonized us in completing this work and for fostering an excellent academic atmosphere.
We are grateful to Dr. S Murthy Shekhar, Professor and Head, Department of Chemical Engineering, SIT, Tumakuru for giving us support throughout this dissertation work.
We express our immense gratitude to our guide, Dr. Thomas Theodore, Assistant Professor Department of Chemical Engineering, SIT, Tumakuru for his guidance, his enthusiastic encouragement that has been a tremendous assistance to us and for his valuable suggestions.
We are also thankful to our co-guide, Dr. Lalithamba, Assistant Professor and UMA K, Department of Chemistry, SIT, Tumakuru for her guidance, support, advice and his valuable suggestions.
Finally, we would like to thank all the teaching and non-teaching staff members of Department of Chemical Engineering for their invaluable cooperation.
SANKET W K
We hereby declare that this dissertation work entitled ‘DIRECT CONVERSION OF N-PROTECTED AMINO ALCOHOLS INTO AMINO ALKYL AZIDES AND SYNTHESIS OF TRIAZOLES’ is carried out by us under the guidance of Dr. Thomas Theodore, Department of Chemical Engineering, and co-guide by Rr.Lalithamba, Assistant professor,Department of Chemistry, SIT, Tumakuru for fulfilment of the academic requirement for the Eighth semester of Bachelor of Engineering B.E for the academic year 2017-18.
We also declare that, the matter contained in the project has not been submitted elsewhere for the award of any Degree or Diploma.
SANKET W K
Azide is one of the most versatile functional groups in organic synthesis owing to the fact that it is the most convenient source of amines, which are very common in natural products as well as pharmaceutical heterocycles.
The stability of azides under physiological conditions and their unique reactivity patterns make them one of the most preferred functionalities in click chemistry. N-protected amino alkyl azides are used in the synthesis of triazole residue, which has been inserted as a crucial structural motif in antimicrobial, anti-HIV and anticancer reagents. They are also useful as inhibitors against tuberculosis, dyestuffs, photo stabilizers and they also find applications in pharmaceuticals and agrochemicals industries.An efficient method for the synthesis of N?-protected amino alkyl azides employing 2-azido-1,3-dimethyl imidazolinium hexafluorophosphate (ADMP) and 4-dimethylaminopyridine (DMAP) has been achieved.N?-protected amino alcohols were treated with ADMP, to yield the desired compounds in excellent yields.
Further, the alkyl azides were converted into N? -protected 1,4-Substituted 1,2,3-triazoles. The synthesized compounds were mainly solids and few were gums after the simple workup, and were characterizedby IR, 1H NMR, 13C NMR and HRMS. Antibacterial activities have been conducted for the few of the synthesized alkyl azides.
Peptide bind formation
Amide bond formation – methods and strategies
N – terminal protecting groups
Fluorenylmethyloxycarbonyl chloride (Fmoc – Cl)
Carboxybenzyl (Cbz) protecting group
Di-tert-butyl decarbonate protecting group
Alloc protecting group
Lithographic protecting groups
Side-chain protecting group
N-protected amino alcohols
Applications of N-protected Amino Alcohols
Synthesis of N-protected Amino Alcohols
Coupling reagent method (one-pot synthesis)
Mechanism of action
Materials and methods
Chemicals and reagents used
Preparation of F-moc protected amino acid
Preparation of N-protected amino alcohols by mixed anhydride method
Analysis of samples by high resolution mass spectroscopy (HRMS)
Working principle of HRMS
Analysis of samples by proton NMR spectroscopy
Analysis of samples by 13C NMR spectroscopy
Analysis of samples by TLC
Results and discussions
1.1 Peptides and peptideomimetics
Peptides and peptideomimetics are ubiquitous in nature and play a key role in numerous biological and physiological made of amino acids that are linked with peptide bonds. They are involved as hormones and neurotransmitters in intercellular communication, antibodies in the immune system, and also in the transport of various substances through biological membranes. The unique features of amide backbone are utilized for effective bonding with receptors to accomplish drug action However, natural peptides possess a few drawbacks such as poor solubility and low potency to pass through cell membrane which are detrimental for their use as drugs effectively. The focus on improving the utility of peptides ass drugs by modifying the peptide backbone and introducing unnatural linkages has lead to a variety of peptidomimetics.
Peptides are a rich source of pharmacophores from which medicinal chemists are developing new useful therapeutic drugs. After binding to an enzyme, or a membrane receptor, peptide-based inhibitors, neurotransmitters, immune modulators (the active agents of immunotherapy are collectively called immunomodulators). These are the agents that augment or diminish immune responses and hormones influencing cell to cell communications and control a variety of vital functions such as metabolism, immune defence, digestion, respiration, etc
Though naturally occurring peptides based on coded amino acids have been widely used as drugs, there are problems with the use of peptides as therapeutic agents. The problems mainly arise from their raoid respiration metabolism by proteolysis and their interactions at multiple receptors. As a results, peptide researchers have sought modifications of peptide structure to create more stable molecules.
Peptides have found wide applications as bio-stable, bio-available, and often potent surrogates of naturally-occuring peptides. They form the basis of important families of enzyme inhibitors and act as receptors agonists and antagonists.
A peptidmimetic is a compound that is designed to mimic a biologically-active peptide, but has structural differences that give greater advantages for its function as a drug.
For instance, a peptidomimetic that is designed to mimic a hormone would have greater stability and be its target to transmit signals. Peptidomimetics may have unnatural amino acids or other unusual compounds to stabilize their structure or alter their biological activity.
The reason for the interest in peptides is that many significant biological activity. This means they can act as hormones and signal molecules for the central nervous system and the immune system. Peptides can effect a wide range of cellular activity, among them digestion, reproduction, and sensitivity to pain. Many peptide activities like cellular and biological are of interest as targets for drugs, but it can be difficult for them to cross the membrane to enter the cell. Also, peptides that do make it into a cell are frequently unstable.
A peptidomimetics is a small protein-like chain designed to mimic a peptide. They typically arise either from of an existing peptide or by designing a similar system that mimic peptides, such as peptiods and ?-peptides. Irrespective of the approach, the altered chemical structure is designed to advantageously adjust the molecular properties such as stability or biological activity. This can have a role in the development of drug-like compounds from existing peptides. These modifications involve changes to the peptides that do not occur naturally (such as altered backbones and the incorporation of non-natural amino acid).
Peptidomimetics are molecules predicted to exert a similar biological as natural peptides but with additional advantages such as good oral absorption and metabolic stability. They are molecules in which one or more peptide bonds are replaced by diverse kinds of unnatural linkages such as ureas, carbamates, sulphonamides, azatides, thiourea, acteals, phosphones, and hetrocycles like tetrazoles, triazoles, oxazoles, etc. and have gained remarkable significance in synthetic and medicinal chemistry as substitutes to the native peptide bond.
Reduced amide peptide
Azide is one of the most versatile functional groups in organic synthesis owning to the fact that it is the most convenient source of amines, which are very common in natural products as well as pharmaceutical heterocyles.
Additionally, the stability of azides under physiological conditions and their iminitable reactivity patterns make them one of the most sought-after functionalities in click chemistry as well as in bioconjugation via Straudinger ligation. Therefore, the development of efficient protocols for the synthesis of azides using easily available, yet efficient reagent systems is of immense importance. In spite of the availability of a wide range of indirect methods, the most common being the substitution of alkyl halides with inorganic azides, direct methods for the said transformation are only a few. The most familiar and versatile one is the Mitsunobu displacement, where hydrazoic acid is used as azide source in the presence of diethyl azadicarboxylate (DEAD) and triphenylphosphine as hydroxyl group activator. As hydrazoic acid is considered a health hazard, a few modifications of the Mitsunobu displacement reaction have surfaced, which include the use of diphenylphosphorazidate (DPPA),bis(2.4-dichlorophenyl)phosphate/NaN3,N-(p-toluenesulphonyl)imidazole/NaN3 /Et3N/TBAI. In a bid to replace the expensive DEAD from the Mitsunobureagent, Hendrickson and Hussoin reported the use of triphenylphosphonium anhydride trifluoromethane- sulphonate, which has the advantage of recyclability by treatment with trifluoromethanesulphonic anhydride (Tf2O). But the method suffers from a serious disadvantage of generation of olefins from secondary alcohols.
An efficient alternative to DEAD was reported by Laa at al. in the form of CBr4, where synthesis of azides by the MItsunobu type displacement reaction was carried out using PPh3/CBr4/NaN3. But, in this procedure the separation of unreacted triphenylphosphine from the products is always very tedious. Here we report a practical procedure for the conversion of alcohols to azides in DMSO using NaN3, PPh3, I2, and imidazole at room temperature.
2.1 Peptide bond formation
The most predominantly used method for peptide bond formation is the acid azide method, introduced by Theodor Curtius more than a century ago. In spite of many newer methos, the Min reason for the continued use of the acid azide route is its ability in yielding optically pure products.
Acyl azides in general and N-protected a-amino acid azides in particular, have occupied a place of their importance in organic, peptide as well as peptidomimetic synthesis. The total synthesis of bovine pancreatic ribonuclease (RNase) A with 124 amino acid was accomplished by consecutive assembly of 30 peptide fragments employing the acid method by Haruaki Yajima. Bodanszky et al., explored the utility of the azide method for the synthesis of secretin. Merrifie;d demonstrated the usefulness of amino acid azides in the stepwise synthesis of octapeptide oxytocin by a solid-phase method polystyrene resin.
Amino acid azides are extensively used in the preparation of amides and peptides and a wide range of other compounds such as nitriles and several classes of hetrocycles. More prominently, they have been utilizing as essential intermideates for the synthesus of gem-dia,imoalkylamines, oligo ureas, etc. under carefully controlled conditions, the acid azide method allows recimization only at a very low level and thus it is routinely used in the segment condensation of peptide fragments.
Acyl azides are usually prepared from the reaction of acid chlorides or mixed with an azide ion(figure 2.1). the acid chlorides are prepared by treating acids with SOCl2. However, this method offers disadvantage for preparation of acid chloride itself. This include prolonged reaction duration, incompatibility with the acid sensitive groups, and storage and stability problems associated with moisture sensitive acid chlorides. Also, the insolubility of NaN3 in organic solvents require the usage of phase transfer catalyst or other catalyst such as ZnI to improve the yield of acid azides. Preparation of acid azides via mixed anhydrades has been used to the advantage. At, this method involves the use of chloroformates which are inconvenient for handling.
2.1.1 Peptide Synthesis
In nature, peptide synthesis involving a sequence of peptide coupling reactions is very complex, probably to safe guard the unique and precisely defined amino acid sequence of every protein. This barrier is overcome in-vivo by a selective activation process catalysed by enzymes, where the required amino acids is transformed into an intermediate amino esther. This intermediate is then involved in a process mediated by the co-ordinated interplay of more than a hundred macro molecules, including m-RNAs, t-RNAs activating enzymes, and protein factors, in addition to ribosomes.
2.1.2 Amide Bond Formation- Methods and Strategies
Carboxyl components can be activated as acyl halides, acyl azides, acyl imidazoles, anhydrates, esthers, etc. there are different ways of coupling reactive carboxy derivates with an amine;
An intermediate acylating agent formed is isolated and subjected to amino lysis
A reactive acylating agent is formed from the acid in a separate step(s), followed by immediate treatment with the amide.
The acylating agent is generated in-cito from the acid in the presence of the amine by the addition of an activating or coupling agent.
2.2 Protecting Groups
Amino acids have reactive moieties at the N- and C-termini, which facilitates amino acids coupling during synthesis. Many amino acids also have reactive sites and functional groups which can interact with the free termini or other side chain groups during synthesis and peptide elongation and negatively influence yield and purity. To facilitate proper amino acid synthesis with minimal side chain reactivity, chemical groups have been developed to bind to specific amino acids functional groups and blocks, or protect, the functional group, from the non specific reactions. This protecting groups, while vast in nature, can be separated into three groups, as follows:
N-terminal protecting groups
C-terminal protecting groups
Side-chain protecting groups
A protecting or protective group is introduced into a molecule by the chemical modification of a functional group in order to obtain chemo selectivity in a subsequent chemical reaction. It plays an important role in multi-step organic synthesis.
In many preparation of delicate organic compounds, some specific parts of the molecules cannot survive the required reagents or chemical environment. Then, these parts, or groups, must be protected. For example, lithium aluminium hydride is a highly reactive reagent but is a very much useful reagent capable of reducing esters to alcohols. It always reacts with carbonyl groups and this cannot be discouraged by any means. When the reduction of an ester is required in the presence of carbonyl, the attack of the hydride on the carbonyl must be prevented. For example, the carbonyl is converted into an acetal, which does not react with hydrides. The acetal is then called as an protecting group for the carbonyl. After the step involving the hydride is complete, the acetal is removed, giving back the original carbonyl. This step is called deprotection.
Protecting groups are more commonly used in small scale laboratory work and initial development than in industrial production processes because their use adds additional steps and material costs in the processes. However, the availability of the cheap chiral building blocks can overcome this additional cost.
2.2.1 N-terminal protecting groups
Amino acids are added in excess to ensure complete coupling during each synthesis steps and without N-Teminal protection, polymerization of unprotected amino acids could occur, resulting in low peptide yield or synthesis failure. N-teminal protection requires an additional step of removing the protecting group, termed deprotection, prior to the coupling step, creating a repeating design flow as follows;
Protecting group is removed from the amino acid in the deprotection reaction
Deprotection reagents are washed away to provide a clean coupling environment
Protected amino acids dissolved in a solvent such as dimethyl formamide(DMF) combined with coupling reagents are pumped through the synthesis column
Coupling reagents are washed away to provide clean deprotection envirornment
Currently, two protecting groups t-Boc, F-moc are commonly used in soilid phase peptide synthesis. Their liability is caused by the carbamite group which readily releases CO2 for an irreversible decoupling step.
Some of the commonly used protecting groups are as follows;
Carboxybenzyl(CBZ) protecting group
Di-tert-butyldicarbonate protecting group
Alloc protecting group
Lithographic protecting groups
2.2.2 Fluorenylmethyloxycarbonil chloride (Fmoc-Cl)
Fmoc-Cl is a chloro formate esther it is used to introduce fmoc groups in the form of fmoc carbonate. This compound may be prepared by reacting Fluorenylmethanol with phosgene
Fmoc protection has found significant use in solid phase peptide synthesis because its removal with piperidina solution does not disturb the acid labile linker between the peptide and the resin. Because the fluoremyl group is highly fluorescent, certain UV inactive compounds may be reacted to give the fmoc derivates, suitable for analysis by reversed-phase HPLC.
Analyticaluses of Fmoc-Cl that donot use chromatography may be limited by the requirement that the excess fmoc-Cl be removed before an analysis of the fluorescence.
2.2.3 Carboxybenzyl (Cbz) protecting group
Carboxybezyl(abbrivated as Cbz or Z) is a carbamide which is often used as an amine protecting group in organic synthesis it is commonly used in peptide synthesis where carboxybezyl protection group is introduced by reacting the amine functionality with benzyl chloroformate in the presence of a weak base.
2.2.4 Di-tert-butyldicarbonate protecting group
Di-tert-butyldicarbonate is a reagent widely used in organic synthesis this carbonate esther reacts with amides to give n-tert-butoxycarbonyl or so called t-Boc derivates this derivates donot behave as amines, which allow certain subsequent transformations to occur that would have otherwise affected the amine functional groups, the t-Boc csn later be removed from the amines using acids. T-Boc serves as a protective group, for instance in solid phase peptide synthesis. It is unreactive to most bases and nucleophiles, amine for an orthogonal f-moc protection. Di-tert-butyldicarbonate is inexpensive, so it is usually purchased. Classicaly, this compound is prepared from tert-butanol, CO2 and phosgene using DABCO as a base.
2.2.5 Alloc protecting group
The allyl oxy carbonyl(alloc) protecting group is often used to protect a carboxylic acid, hydroxy, or amino group when an orthogonal deprotection scheme is required. It is some times used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side chain functional group. The alloc group can be removed using tetrakis(tri phenyl phophine) palladium(0) along with 37:2:1 mixture of methylene chloride, acetic acid, and m-methylmorpholin(NMM) for 2 hours.
2.2.6 Lithographic Protecting Groups
They are used for special applications like protein micro arrays. These groups can be removed by exposure to light.
2.2.7 Side Chain Protecting Groups
Amino acid side chain represents a broad range of functional groups and are sides of nonspecific reactivity during peptide synthesis. Because of this many different protecting groups are required that are usually based on the enzyme and (Bzl) or tert-butyl (tBa) groups. The specific protecting groups used during the synthesis of a given peptide vary depending on the peptide sequence and the type of N-terminal protection used.
Side chain protecting groups are known as permeant or semi-permanent protecting groups because they can with stand the multiple cycle’s of chemical treatments during synthesis and are only removed during treatment with strong acids after the peptide synthesis is over.
2.3 Protection Scheme
Multiple protecting groups are normally used during peptide synthesis, this groups must be compatible to allow deprotection of distinct protecting groups while not affecting other protecting groups. Protection schemes are therefore established to match the protecting groups so that deprotection of one protecting group does not affect the binding of the other groups. Because N-terminal deprotection occurs continuously during peptide synthesis, protecting schemes have been established in which the different types of side chain protecting groups (Bzl or tBu) are matched to either Boc or Fmoc, respectively, for optimized deprotection.
2.4 Activating Groups
For coupling the peptides, the carboxyl group is usually activated. This is important for the speeding of the reaction. There are two main types of activating groups: cabodiimides triazolols. However, the use of pentyafluorophenyl esters is useful fpr cyclizing peptides.
This activating agents were developed first and the most common amog them are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Reaction with a carboxylic acid is a highly reactive O-acylisourea. During artificial protein synthesis (such as Fmoc solid- state synthesizers), the C-terminus is often used as the attachment site on which the amino acid monomers are added to enhance the electrophilicity of carboxylate, negatively charged oxygen must first be invited” activated” to a better living group. DCC is uised for this purpose. The negatively charged oxygen will act as a nucleophile, attacking the central cabon in DCC. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiimides is that they are too reactive and can therefore cause racemization of the amino acid.
2.5 N-protected amino alcohols
N-protected amino alcohols contain both an amine and an alcohols group. Amino alcohols derivatives have been employed as catalysts as well as coupling partners in the synthesis of many compounds. Enantiomerically pure ?-amino alcohols play an increasingly important role in pharmaceutical therapy and as chiral auxiliaries in organic synthesis. Amino alcohol derivatives are currently being studied for their antimicrobial and antifungal activities and in the modulation of the physiological properties of the drug molecules.
The amino alcohol group is present in several antibiotics, such as ethambutol for the treatment of tuberculosis. 1,2-addtions, ring-closure reactions, conjugate additions, and ?-functionalization of carbonyl compounds are effectively accomplished by ?-amino alcohols as catalysts. The ready availability of ?-amino alcohols from a chiral pool (e.g., L-amino acids) makes them appealing class of versatile promotors to exploit in modern organic synthesis.
2.6 Applications of N-protected Amino Alcohols
N-protected Amino Alcohols and peptidyl alcohols are used in the synthesis of peptide-bonds surrogates as key intermediates and also in synthesis of amino peptidyl aldehydes and stereochemically-defined methylene-oxy dipeptides.
N-protected Amino Alcohols are used in the asymmetric synthesis, in the synthesis of insecticidal compounds, peptide possessing reduced amide bonds, and in the preparation of the receptors for carbohydrate recognition.
N-protected Amino Alcohols are versatile building blocks in organic synthesis.
N-protected Amino Alcohols acts as a precursor and building block for many compounds.
N-protected Amino Alcohols are used as building blocks for synthesis of HIV protease inhibitors.
2.7 Synthesis of N-protected Amino Alcohols
There are several protocols available for the synthesis of N-protected Amino Alcohols; among these, the coupling reagent method and the mixed anhydride method have found widespread application in the field of peptide synthesis.
2.7.1 Coupling reagent method (one-pot synthesis)
Amino acids such as those derived from the Boc and Z-protected ?-amino acids are not relatively stable to the extent of isolation. It is desirable to develop one-pot protocols which involve tandem transformation of in situ generated acyl azides into the target molecules. Such protocols are also highly useful for the rapid synthesis of a library of biologically-active analogs for screening.
An example of this is the one-pot synthesis of amino and peptidyl alcohols starting from acids which involves the formation of acid azides and their in situ Curtius rearrangement followed by coupling of isocyanates with amines and alcohols. The procedure for generating acid azides using NaN3 and chloroformates or Boc2O, and the rearrangement and trapping of the isocyanates in the presence of n-Bu4NBr and zinc triflate, have been reported. A continuous flow reactor, designed for the sequential rearrangement of acid azides and coupling of isocyanates, has also been used to prepare ureas and carbamates. DPPA and Deoxo-Flour/TMS-N3 have been employed in the preparation of peptidyl and sugar ureas. However, the application of peptide-coupling agents for this purpose has not been described earlier. Peptide synthesis relies heavily on efficient and reliable coupling reagents. A low tendency for racemization is a key requirement. This is especially true for solid phase peptide synthesis-quantitative yields with short reaction times are of utmost importance to make the synthesis of large peptides feasible.
A plethora of methods for the formation of peptide bond have been reported. The most successful approaches known today involve active ester formation with uranium/guanidinium salts. The most popular members of this family are peptide synthesis reagents based on benzotriazole derivatives such as HOBt or HOAt, both of which are also commonly used as additives in carbodiimide-mediated peptide coupling.
To solve the problem of racemization, triazoles were introduced. The most important ones are 1-hydroxy-benzotriazole and 1-hydroxy-7-aza-benzotriazole. These substances can react with the O-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favourable because of a neighbouring group effect. Recently, HOBt has been removed from many chemical vendor catalogue; although almost always found as a hydrate, HOBt can be explosive when allowed to fully dehydrate and shipment by air or sea is heavily restricted. Alternatives to HOBt and HOAt have been introduced. One of the most promising and inexpensive is ethyl 2-cyano-2-(hydroxyamino) acetate (trade name Oxyma Pure), which is not explosive and has a reactivity of that in between HOBt and HOAt.
2.9 Mechanism of action
Direct transformation of alcohols to azides is an attractive and efficient strategy for the synthesis of alkyl azides. In the present work, it is desired to propose an efficient protocol (Figure: ) for the synthesis of N?-protected ?-amino alkyl azides in excellent yields from their corresponding protected alcohol by stirring a solution of sodium azide in DMSO with thoroughly ground equimolecular mixture of triphenylphosphine, iodine, and imidazole. This method is easy, fast, and free from racemization. After the usual work-up and column purification, the obtained products will be fully characterized by IR, 1H, 13C NMR, and Mass spectral studies.
Synthesis of N?-protected ?-amino alkyl azides
Materials and Methods
3.1 Experimental setup
The experimental setup consists of magnetic stirrer, round-bottomed flask, guard tube, and magnetic pellet. The protected amino acid sample is placed inside the round-bottomed flask and stirred with the help of magnetic stirrer. Temperature of about 0 0C is maintained by placing ice around the round-bottomed flask.(Figure 3.1).
3.2 Equipment’s used
3.3 Chemicals and reagents used
Amino acids (Phenyl alanine, alanine, valine, lucine, isoleucine)
Sodium borohydride (NaBH4)
Sodium azide (NaN3)
A known quantity of amino acid sample is taken in around-bottomed flask and dissolved using THF and stirred using magnetic stirrer for 2 min. the temperature of round-bottomed flak is maintained around 0 0C. A calculated amount of NMM is added followed by ECF. The setup is left for half an hour. The contents are filtered and washed with THFD. A known quantity of NaBH4 is added, stirred for 2 min and kept in a water bath for 30 min. the dry solid is collected and weighed. Conversion of amini acid to alcohol is confirmed by thin layer chromatography (TLC).
Amixture of Fmoc alcohol, triphenylphosphine, imidazole and iodine was ground in a morter with a pestle for 10 min top make a paste with an exothermic reaction took place. Then a solution of sodium azide in DMSO was added and stirred for 30 min. Upon completion of the reaction, the mixture was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and conc. Under vaccum to get a crude product which was purified by column chromatography using 5% ethyl acetate in hexane as eluent to get the final product.
Figure No : Formation of alcohol
3.4.1 Preparation of Fmoc-protected amino acid
A clean and dry 100-ml round-bottomed flask was placed on a magnetic stirrer with a magnetic pellet inside it. 200 mg of amino acid and 2 g of sodium carbonate weighed and transferred to the round-bottomed flask. 20 ml of water also added to the flask. To this mixture, solvent dioxane was added with the intermintent addition of Fmoc. Dioxane was mainly added to dissolve the acid. The reaction was allowed to proceed at room temperature overnight after which the formation of the protected acid was checked with the help of TLC plate. The place was eluted with CMA (40:2:1) in an elution chamber until the 80% of the TLC plate was eluted. It was then kept inside a UV-Chamber. The formation of the protected acid was confirmed by the deposition of a spot on the TLC plate.
3.4.2 Preparation of N-protected amino alcohols by mixed anhydride method
This protocol describes the preparation of amino alcohols by the mixed anhydride method. This is based on the earlier protocol used for the production of protected amino alcohols. 200 mg of prepared amino acids prepared was taken in a 100-ml round-bottomed flask and 0.5 ml of N-methylmorphiline and ethyl chloroformate were added to it. The amino acid reacts with the alkyl chloroformate in the presence of based form carbonic acid anhydride which was further dissolved in a few drops of tetrahydrofuran. To the above mixture, a known quantity of sodium borohydrate that has been calculated for particular amino acid was added and the reaction was allowed to proceed to -15 deg.C for 10 to 15 min. the protected alcohol was concentrated in a roto-evaporator. The reaction mixture may contain many acidic and basic impurities and workup is needed to obtain a pure product. The prepared sample was tested on TLC plate to confirm the formation of amino alcohol. Three spots were chosen on TLC plate. The first spot represents the amino acid, the second spot represents the reaction mixture and the third spot represented the amino alcohol. The sample to be analysed was eluted in 40% concentration of ethyl acetate-hexane mixture before introducing into the UV-Chamber. The formation of amino alcohol was confirmed by observing the movement of spot under UV light. Only the reaction mixture and alcohol spot move under UV light, where as the amino acid retains its position on the TLC plate. If the spots are too faint, TLC plates are kept inside an iodine chamber and then analysed.
3.5 Analysis of samples by high resolution mass spectrometry(HRMS)
Mass spectrometry is an analytical technique that bprodueces spectra of the masses of the atoms or molecules comprising a smaple of material. The traditional anmd continuing justification for high resolution mass spectrometry(HRMS) is for the identification or confirmation of the molecular formulas of new compounds. In this study, HRMS measurements are used to accurately determine the mass of the molecular ion. These structural applications of HRMS were first employed for organic compounds but have now been adapted to other areas of chemistry as well, e.g, to the characterisation of organo metallic and inorganic compounds and more recently to the study of bio-polymers. A general scheme for using mass spectrometry to identify an unknown compound is as follows:
Determination of the molecular mass of the compounds from the mass spectrum, ideally the highest m/z ratio ion in the mass spectrum corresponds to an isotropic form of the intact ionic molecule.
Identification of the functional groups present in the molecule on the basis of specific fragments ions and/ or fragment ion series contained in the mass spectrum characteristic fragment ions formed by the loss of the neutral molecules from the intact ionic molecule that are characteristic of specific groups.
Assemblage of the identified functional groups for the prediction of the structure of the molecule. An early example of the analytical utility of HRMS was demonstrated for the detailed characterisation of the different high boiling fraction of petroleum in terms of functional group composition.
3.5.1 working principal of HRMS
High resolution mass spectrometry(HRMS) provides information pertaining to the molecular weight, elemental composition and molecular structure of a compound. HRMS works by ionising chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge(m/z) ratio.
As shown in figure 3.3, a mass spectrometer consists of three components: an ion source, a mass analyzer and a detector. The ionizer converts portion of the sample into ions. There is a wide variety of ionisation techniques, depending on the phase(solid, liquid or gas) of the sample and the efficiency of various ionization mechanisms available for the unknown species. An extraction system removes the ions from the samples and directed through the mass analyzer and on to the detector. The differences in mass of the fragments allowed the mass analyzer to sort the ions according to their mass-to-charge(m/z) ratio. The detector measures the value of an indicator quantity and thus provides data for calculating the abundance of each ion present. Some detectors also give spatial information.
3.6 Analysis of samples by proton NMR spectroscopy
Proton NMR (hydrogen-1 NMR, 1H NMR) is the application of nuclear magnetic resonance (NMR) spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance in order to determine the structure of its molecules. In samples where natural hydrogen (H) is used, practically all of the hydrogen consists of the isotope 1H (hydrogen-1, i.e., having a proton for a nucleus). A full 1H atom is called protium.
Simple NMR spectra are recorded in solution and solvent protons must not be allowed to interfere. Deuterated (deuterium = 2H, often designated as D) solvents are preferred for use in NMR, e.g., deuterated water(D2O), deuterated acetone ((CD3)2CO), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide ((CD3)2SO), and deuteratedchloroform (CDCl3). However, a solvent without hydrogen such as carbon tetrachloride (CCl4) or carbon disulphide (CS2) may also be used.
Proton NMR spectra of most organic compounds are characterized by chemical shifts in the range +14 to – 4 ppm and by spin-spin coupling between protons. The integration curve for each proton reflects the abundance of the individual protons. Together with carbon-13 NMR, proton NMR is a powerful tool for molecular structure characterization.
Chemical shift values, symbolized by ?, are not precise, but typical and they must therefore be regarded only as a reference. Deviations are in the ±0.2 ppm range and sometimes more. The exact value of the chemical shift depends on the molecular structure, the solvent used, temperature, magnetic field strength, and other neighbouring functional groups. Hydrogen nuclei are sensitive to the hybridization of the atom to which the hydrogen atom is attached and to electronic effects. Nuclei tend to be de-shielded by groups which withdraw electron density. De-shielded nuclei resonate at higher ? values, whereas shielded nuclei resonate at lower ? values.
The chemical shift is not the only indicator used to assign a molecule. Because nuclei themselves possess a small magnetic field, they influence each other, changing the energy and hence the frequency of nearby nuclei as they resonate—this is known as spin-spin coupling.
A peak is split by n identical protons into components whose sizes are in the ratio of the nth row of Pascal’s triangle (Fig3.6):
Figure 3.6Pascal’s triangle
Because the nth row has (n+1) components, this type of splitting is said to follow the “(n+1) rule”: a proton with n neighbours appears as a cluster of n+1 peaks.
Below (Fig 3.7) are represented NMR signals corresponding to several simple multiplets. Note that the outer lines of the nonet (which are only 1/8 as high as those of the second peak) can barely be seen, giving a superficial resemblance to a septet.
Figure 3.7 Corresponding peaks for the (n+1) rule
When a proton is coupled to two different protons, then the coupling constants are likely to be different, and instead of a triplet, a doublet of doublets will be seen. Similarly, if a proton is coupled to two other protons of one type, and a third of another type with a different, smaller coupling constant, then a triplet of doublets is seen. In the example below, the triplet coupling constant is larger than the doublet one.
By convention, the pattern created by the largest coupling constant is indicated first and the splitting patterns of smaller constants are named in turn. In the case below (Fig 3.8), it would be erroneous to refer to the quartet of triplets as a triplet of quartets. The analysis of such multiplets (which can be much more complex than the ones shown here) provides important clues to the structure of the molecule being studied.
The simple rules for the spin-spin splitting of NMR signals described above apply only if the chemical shifts of the coupling partners are substantially larger than the coupling constants between them. Otherwise, there may be more peaks and the intensities of the individual peaks will be distorted (second-order effects).
3.7 Analysis of samples by 13C NMR spectroscopy
Carbon-13 NMR (13C NMR or sometimes simply referred to as carbon NMR) is the application of nuclear magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR (1H NMR) and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. As such 13C NMR is an important tool in chemical structure elucidation in organic chemistry. 13C NMR detects only the 13C isotope of carbon, whose natural abundance is only 1.1%, because the main carbon isotope, 12C, is not detectable by NMR since it has zero net spin. 13C NMR has a number of complications that are not encountered in proton NMR. 13C NMR is much less sensitive to carbon than 1H NMR is to hydrogen since the major isotope of carbon, the 12C isotope, has a spin quantum number of zero and so is not magnetically active and therefore not detectable by NMR. Only the much less common 13C isotope is magnetically active with a spin quantum number of 1/2 (like 1H) and therefore detectable by NMR. Therefore, only a few 13C nuclei are present in the magnetic field, although this can be overcome by isotopic enrichment of the protein samples.
In further contrast to 1H NMR, the intensities of the signals are not normally proportional to the number of equivalent 13C atoms and are instead strongly dependent on the number of surrounding spins (typically 1H). The spectrum can be made more quantitative if necessary by allowing sufficient time for the nuclei to relax between repeat scans. The 13C chemical shifts follow the same principles as those of 1H, although the typical range of chemical shifts is much larger than for 1H (by a factor of about 20). The chemical shift reference standard for 13C is the carbons in tetramethylsilane (TMS), whose chemical shift is considered to be 0.0 ppm.
3.8 Analysis of samples by TLC
A TLC plate is a sheet of glass, metal or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analysed is spotted near the bottom of the plate. The TLC plate is then placed in a shallow pool of the solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This solvent rises up the TLC plate by capillary action. As the solvent passes the spot that was applied, equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber and the separated components of the mixture are visualized in a UV chamber as shown in Fig 3.10. The plate itself contains a fluorescent dye which glows everywhere except where an organic compound is on the plate.
Figure 3.10 TLC plate showing the movement of spots under UV light
The solvents used for diazomethylketone and bromomethylketonesyntheses were purifiedas follows:
Distilled over P2O5and stored in a brown-coloured bottle over molecular sieves.
Kept over anhydrous CaCl2 overnight, decanted, refluxed over sodium wire for 1 hour, distilled and finally stored over sodium wire.
Shaken well with CaO, decanted and distilled.
Treated with K2CO3 overnight, filtered and distilled over P2O5.
Refluxed over magnesium turnings (5 g/L) in the presence of traces of iodine and distilled.
Shaken with KOH pellets for 2 hours, decanted, refluxed over sodium wire in the presence of benzophenone till the solution became blue, distilledand stored over sodium wire.
RESULT AND DISCUSSION
The samples of Fmoc-Ala-Triazole was prepared and characterized by the fallowing techniques:
Proton NMR Spectroscopy
Carbon-13 NMR Spectroscopy
In the present work, the target protected triazoles were prepared from N-protected amino acids. Fmoc-amino acids were converted to the corresponding ?-amino alcohols. An efficient method for the synthesis of N?-protected amino alkyl azides employing 2-azido-1,3-dimethyl imidazolinium hexafluorophosphate (ADMP) 4-dimethylaminopyridine (DMAP) has been achieved.N?-protected amino alcohols were treated with ADMP, to yield the desired compounds in excellent yields. Further, the alkyl azides were converted into N? -protected 1,4-Substituted 1,2,3-triazoles.
The Synthesized compoundsare characterized by IR ,1HNMR ,13CNMR and HRMS Few of the synthesized compounds were subjected to biological activities.
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