Structural Components Of Plasma Membrane


INTRODUCTION

Cell is a basic structural and functional unit of living organisms. Cells are the building blocks of life. Cell consists of protoplasm and nucleus. It is surrounded by a cell membrane (plasma membrane) that protects it from external environment and acts as a barrier. It is almost identical to the membranes that surround nucleus and other organelles. The membrane is made up of protein-lipid bilayer consists of a polar head and a non-polar tail. The fluidity of the membrane is regulated on the basis of lipid content and responsible for proper functioning of membrane proteins. It plays a vital role in the structure and function of the cell.

The membrane is permeable to specific molecules based on the polarity of the substrate. The structure of the membrane is unique as it exhibits specific protein-lipid content depending upon the location of the membrane. The various biological barriers exists in humans include plasma membrane, blood brain barrier, blood placental barrier etc..,

Structural components of Plasma Membrane:

Membranes are made up of phospholipids, proteins and carbohydrates.

The phospholipids form a flat surface on which proteins span or float like icebergs and carbohydrates protrude out from the phospholipid layer.


The phospholipids are arranged in a bilayer such a way that polar heads faces outside and non-polar tails in the middle of the surface giving a sandwich like appearance. The fatty acids are linked together by cholesterol that strengthens the membrane.
The proteins usually exits in two forms, one spanning the surface as integral proteins and other as on the surfaces of the membranes as peripheral proteins. They are responsible for majority of the membrane properties.
' They exist as transport vehicle that causes exchange of substance across the cell.
' They act as receptors and enzymes catalyzing reactions in cytoplasm.
' They involve in cell signaling, cell-cell recognition and maintain cell`s shape.
The carbohydrates are found on the outer surface of all eukaryotic cell membranes, and are attached to the membrane proteins or sometimes to the phospholipids. Proteins with carbohydrates attached are called glycoproteins, while phospholipids with carbohydrates attached are called glycolipids. The carbohydrates are short polysaccharides composed of a variety of different monosaccharides, and form a cell coat or glycocalyx outside the cell membrane. The glycocalyx is involved in protection and cell recognition, and antigens such as the ABO antigens on blood cells are usually cell-surface glycoproteins.
The membranes provide compartmentalization and are vital for the sustenance of life as they are responsible for maintenance of concentration gradient, movement, adherence, and protection.
Transport of substances across the membrane:
The plasma membrane is selectively permeable that some substances can pass and some cannot. Ions and charged molecules cannot enter because they are unable to cross lipid layer and uncharged substances can pass through it.
Usually they follow the concentration gradient across the membrane and thus does not require energy hence a passive process. The movement across the membrane can be classified in to Active process and Passive process.

a) Active process:
The molecules are transported by carrier proteins that span the membranes they utilize the energy in the form of ATP hence active process. A carrier protein acts as a pump that initiates a substance against the concentration gradient.
b) Passive process:
A carrier protein assists molecules to move along the concentration gradient. No energy is required.

Fig.2. Transport of molecules across the membrane
Summary of Membrane Transport:
Process Uses energy Use proteins Controllable Specific
Diffusion No No No No
Osmosis No No Yes No
Passive transport No Yes Yes Yes
Active transport Yes Yes Yes Yes
Vesicles Yes No No Yes

As the plasma membrane is impermeable to many of substances the physiological system has developed some intrinsic transport vehicles in the form of membrane transporters that are selective for specific molecules.

DRUG DELIVERY ' A CHALLENGE
The pharmacotherapy aims to treat the disease at molecular level where the desired drug has to reach site of action at minimal concentration. Many bioactive agents are polar in nature thus unable to cross the barrier. The therapeutic efficiency of a drug depends on the ability to reach the site of action. The major obstacle for pharmaceutical agents(drugs, peptides, oligonucleotides, plasmids) that show promising activities in vitro fails to act in vivo due to various pharmacokinetic and bioavailability problems. The ability of a drug to cross such biological barrier depends on its intrinsic physical property. To enhance the activity one should enhance the efficiency of cell transfection across the membrane. Intracellular delivery of therapeutic agents is one of the challenges in drug delivery in case of gene therapy which now a days considered as a major contender in future treatment protocols for threatening diseases like cancer and AIDS. Efficient delivery of therapeutic and diagnostic agents across cell membrane is crucial for developing novel therapies.

To achieve maximum therapeutic efficiency many pharmacologically active proteins and peptides has to be delivered intra-cellulary at subcellular level to modulate its functions.
A conventional method of delivering genetic material is using viral vectors, however confined to limited success. Alternative non-viral methods such as electroporation, microinjection and the use of liposomes are proved to be effective invitro and show limited potential invivo due to toxicity, cell damage and immunogenicity.

Historically drug design and selection is based on the Lipinski rule as it states only compounds possessing certain log P range are efficient and able to cross the lipid membrane and other substances cannot. However all the drug candidates with effective log P are not promising agents and suffer various pharmacokinetic and pharmacodynamics problems.

Many polar and some non-polar molecules possess good therapeutic potential are facing difficulty in cell transfection. To address these problems an approach that modulate the physical properties of a substance that exhibit poor bioavailability, and suffers from distribution, metabolism and excretion is to tether the agent to a molecular transporter.

MOLECULAR TRANSPORTER:
'Molecular Transporters are the agents, when attached to a poorly bioavailable drug, drug candidate or a probe that exhibit excellent water solubility and simultaneously potentiates its passage through biological barriers'.
The term applies to all structural varities of agents that enable the passage of substances across membrane. The goal of enhancing the delivery of cargo is to potentiate its water solubility and cellular uptake. Molecular transporters are capable of specifically delivering a cargo targeted to a tissue or organ.
The cargo tethered to the carrier has to be cleaved after cellular entry either by abiological methods such as due to light, PH, heat or by biological activation by proteases, esterases and redox reactions.
The discovery of natural and synthetic cell penetrating agents has opened new possibilities in biomedical research to potentiate cellular drug delivery.
A vast increase in the use of protein transduction domain technique to combat the problem of introduction of biologically active molecules invivo. Protein transduction domains (PTD) are capable of transducing the cargo across the barrier. This process of protein transduction was discovered independently by Green and Frankel. The mechanisms of transduction are currently unidentified and it is independent of receptors and endocytosis. The electrostatic interaction between opposite charges play important role in protein transduction. The most impressive aspect of PTD was its size independence.
Cell Penetrating Peptides: A Conspectus
The different types of synthetic molecular transporters are peptides, peptoids, Oligocabamates, b-peptides and peptide nucleic acids.
To the surprise Frenkel and Green found that trans activating factor (TAT) was efficiently taken up by the surrounding cells when added to the media. The internalization of TAT, antennapedia and transportan was thought to follow non-saturable, dose dependent kinetics.
The assumption that positively charged residues in the carrier has a critical role in the translocation. The cationic nature of TAT plays a specific role in translocation process. The concept of cell penetrating peptides (CPP) has emerged from serendipitous observations by virologists on the HIV-1 Tat trans-activating factor.
Criterion should be satisfied to act as a cell penetrating agent:
' Small and simple to synthesize
' Able to attach to different cargoes without losing its translocation property
' Should be non-toxic
' Cell, tissue and compartment selective.
The oligomers of arginine alone or when conjugated to other large biomolecules readily cross the plasma membrane. Because of high cationic nature of TAT several anionic substances can influence the cell surface binding such as heparin sulfate, dextran sulfate etc. Membrane adhesion occurs at any temperature and it is an energy dependent process.
Role of heparin sulfate proteoglycans (HSPG):
Cell surface heparin proteoglycans are responsible for the entry of peptides as it acts as receptor for peptides and regulates uptake in time- concentration- and temperature dependent. Heparin sulfate lies on the surface of the membrane and it is the major component in the extracellular matrices. The protein-HSPG interactions enhance activities specific to organs in mammals. The function depends on the core protein interaction.

Fig.3. Role of HSPG in cell physiology

The internalization of transporter require the expression of negatively charged glycosamino- glycans on cell surface for interaction followed by internalization via non-clatharin mediated pathway to lysosomes. The PTD do not originally provoke translocation, but responsible for the adherence which leads to enter in to endosomes.
The number and location of arginine residues in the PTD sequence play a determinant role in the transduction efficiency of a TAT. A significant reduction in the cell penetration is observed when the positive charges are removed. The arginine was replaced with other residues (lysine, ornithine) and with citrulline (having urea linkage) showed completely abolished cell translocation.
Approaches for CPP mediated delivery:
Cell penetrating peptides (CPP) based delivery offer a great potential for cellular delivery of drugs with poor bioavailability. Literature survey reveals that there are four methods for preparing CPP. They include:
A. Recombinant fusion:

The CPP was incorporated in to the therapeutic protein as transduction enhancer as it adds mass to the protein, and fusion product is produced. This method is widely in use for protein conjugates and these are incompatible with polymers, nucleic acids and non-protein moieties.

B. Covalent chemical linkage:

Covalent linkage is extensively used to connect a CPP and cargo. There are various methods in practice such as amide linkage, thiol-maleiimide, thio-ester, and disulphide bond. Mostly a cleavable linkage is preferred to ensure the release of cargo.
C. Non-covalent linkage:
To avoid limitation of the covalent linkage such as strong electrostatic attraction, non-covalent methods are preferred. The complexes are formed via weak electrostatic interactions.

D. Nano particle based carriers:
Incorporation of CPP to the nanoparticle not only enhances the cellular uptake but also the efficiency in delivery of large amount of drugs. As nanoparticle carry variety of drugs with different physical properties those cannot directly connected to the CPP can be loaded here.
Pros and cons of peptide delivery:

OBJECTIVE & PLAN

The plasma membrane is the barrier for various extraneous xenobiotics, many essential large biomolecules are unable to be delivered interior of the cell. Although literature survey reveals that many conventional carriers are reported. They exhibit stability problems and severe limitations in invivo release. Hence there is very important need in development of new carrier molecules that can easily traverse across the membrane and deliver the cargo. Cell penetrating peptides are the agents developed in this regard but they are also avoided due to reasons mentioned above in the text.
The objective of my work describes the need of development of non-peptide based molecular transporter and effective design and synthesis of the transporter.

Plan of work:
A. Design of Non-Peptide Based Molecular Transporter.
B. Synthesis of Designed Transporter.
C. Synthetic Attempts towards More Hindered/flexible Guanidinium Transporter.
D. Cell Transfection Studies of the Transporter in tissue culture.

DESIGN OF NON-PEPTIDE TRANSPORTER:
The peptide based delivery agents/carriers are susceptible to a variety of endogenous proteases in the physiological system, thus limiting their bioavailability. Thus there is strong requirement to develop a non-peptide based molecular transporter in the field of drug delivery with concomitant modifications of physicochemical properties.
The major finding is that the guanidinium group is the key to effectiveness of Tat-inspired transporters. An enhanced cellular uptake is observed with guanidinium analogues compared to amine analogues.

The transfection property is increased with increased number of cationic groups.

Biological Significance of Guanidinium:
The rate of uptake depends on the concentration of the oligomers and guanidine content. The guanidine side chain has PKa of 12 and each residue is highly charged at physiological PH. The NH moiety of guanidine can donate a hydrogen atom thus forms a stable H-bonding with anions present on the surface of the membrane. The positive charges of guanidine moiety needed for efficiency. The characteristic phenomenon of transporter correlates its ability to enter the cell with its guanidine content. The weak acidity of guanidinium hinders partial deprotonation under physiological condition. The intermolecular charge repulsion is minimized by guanidinium oligomer by counter ion scavenging liked to transduction ability.

Fig.5. Complex formed between guanidinium group and phosphate group of plasma membrane through electrostatic interaction

Drugs having guanidinium group:
Natural products possessing guanidinium group:
The ability to enter the cell enhances when the length of side chain, their conformational freedom is increased. The guanidine group forms salt bridges with phosphate or carboxylic ions on surface thus reduces the no of guanidines available for transport.
The intracellular and tissue selectivity can be achieved by varying structural variation in the transporter design such as scaffold, linker and number of guanidinium groups. Uptake is determined by no of guanidinium groups the scaffold does play a role. The spatial orientation of guanidinium can be used for selectivity in performance or localization.
Backbone rigidity:
Chirality is not a crucial factor for uptake as D-oligomer shows better performance compared to natural oligomer due to its greater stability towards proteolysis. Hence the backbone of the transporter is not of critical importance.
The backbone stereochemistry is not essential for uptake opens the possibilities and enhanced the chances of designing various transporter scaffolds even from peptide to non-peptide based systems.
The modified carbamates linkage outperforms by 2.3 times faster than peptide and peptoids transporters and penetrates easily through skin a formidable barrier than cell membrane.
Backbone chirality, type, spacing, H-bonding can be varied but the key is guanidinium group.

Backbones facilitated uptake:


L ' amino acid D - amino acid oligocarbamate

??-peptide 6-aminocaproic acid peptoid

Influence of structural motifs on translocation:
' The peptide backbone and backbone chirality were not essential for uptake.
' The amide linker backbone is crucial for efficient translocation of carbamates.
' The conformational flexibility and steric encumbered nature is important for uptake.
' Changes in backbone composition and side chain spacing increase uptake.
' Highly branched oligomer potentiates cell penetration than linear.
Conformational Flexibility:
The flexibility of oligomer is requisite for enabling contact points between transporter and cargo. Increasing the flexibility of the side chains leads to a higher percentage of guanidine to contact a biological membrane. The enhanced flexibility result in greater efficacy by allowing the molecule to adopt different stable conformations required for various steps in translocation. Both the spatial arrangement of guanidinium groups and the rigidity of the molecular scaffold that displays the guanidinium groups affect the entry of an oligo-cation into live cells.
The increase in flexibility would further increase the percentage of guanidinium capable of interacting with opposite charge on the surface of the membrane.
Two tetra arginines separated by a rigid benzene spacer showed a 3.8 times decrease in uptake suggests that greater the flexibility faster is the uptake.

Summary of structural elements and their role in cell transfection:
Structural element Importance Role in transfection
Primary sequence No Equally effective
Linearity No Branched ones are effective
Positive charges Important Play role in cell surface binding
Chirality No Regardless of D/L form
Guanidine group Important Cell surface adhesion
Side chain length Moderate Longer & more flexible potentiate activity
Peptide backbone No Not essential

Role of membrane potential and H-bonding in translocation:
Charge:
Charge is necessary but not sufficient to cross the barrier, as both ammonium and guanidinium groups possess the charge. As the later forms a bidentate bond it is stronger and possesses ability to associate and traverse cell membrane when compared to former group which forms a single bond. The driving force for the association is the decrease in free energy from solution to surface of the membrane.
The charge itself is necessary but not sufficient for the uptake. This assumption was supported as lysine residues show the poor uptake when replaced with arginine.
The polarity of the guanidinium groups could be attenuated through association with cell surface groups bearing a complementary charge (phospholipids, fatty acid salts, and sulfates), thereby producing a less polar ion pair complex capable of diffusing into the membrane.
The resultant ion pair complexes partition into the lipid bilayer and migrate across at a rate proportional to the membrane potential.
H-bond:
The H-bond formed by the guanidinium group is very crucial for the uptake as it resulted in the reduced ability to penetrate in to cell when the guanidinium group is either methylated or di-methylated. The mono methylated compound showed 95% reduction in cellular translocation and in di methylated the activity completely abolished.

Fig.6.


Fig.7.

Relation between cargo, transporter and cell type:
Wide range of cargo can be transported across a barrier using various molecular transporters but for each cargo, cell type and optimal conditions must be optimized.

SYNTHETIC SCHEME:


Reagents and conditions:
a) Et3N, TrCl3, DMF b) NaBH3CN, (NH4)2 B4O7. 4H2O, MeOH, c) DCM, d) (Boc)2O, DCM e) HgCl2, DIPEA, DMF.
f) AcOH / CF3CH2OH, DCM.

Background:
The guanidinium group has received special attention in organic chemistry, and medicine. It is present in many medicinally important natural products. Guanidinium synthesis has a very rich literature.

Direct guanidylation of amines:

PROPOSED MECHANISMS OF INTERNALIZATION:
The factors such as transporter structure, temperature, incubation time, assay solution, cell type, tissue type, linker and cargo size play a significant role in the internalization of a transporter across a barrier.
The mechanism of translocation involves
1. Association:
The first step of mechanism involves association between transporter and a membrane. Each positive charge forms a rigid H-bond with negatively charged surface of the membrane.
The stronger the association between transporter and a membrane, greater is the chance of internalization. At the same time the association must be reversible.
The association allow for the increased resident time and favours translocation.
2. Mechanism of uptake:

Various cell penetrating agents adopt different mechanisms of uptake into the cell which can be further visualized by confocal microscopy.

a) Endocytosis:

The mechanism of uptake is energy dependent process and cargo sensitive. Endocytosis is widely accepted mechanism of transfection of which most of the studies reveals that clatharin mediated endocytosis is primary mechanism.

Fig.10 Proposed endocytic mechanism of uptake clatharin-, caveolae-, and Macropinocytosis

Macropinocytosis is one of the endocytic mechanism mediated by lipid rafts, and is clatharin-, caveolae-, and receptor independent. The process involves the enclosing of actin- containing membrane protrusions to form vesicles called macropinosomes.

b) Non- endocytosis:

The non- endocytic pathway involves direct diffusion through the membrane. This process would seem to oppose dogma.

c) Inverted micelle model:

Derossi et al. proposed an inverted micelle model in which negatively charged membrane constitutents interacts with positively charged components of the transporter to form an inverted micelle structure that can open on the inside or outside of the cell.

d) Adaptive translocation:

The proposed mechanism involves the strong association of positively charged portions of transporters with the negatively charged constituents of membrane, forming an ion pair complex that probably reduces the polarity of the transporter and able to penetrate into the membrane and further into the cell.

The cellular uptake process maintains a linear relationship with the potassium (K+). Uptake decreases as external [K+] increases and Nernst potential is calculated based on the K+ concentration. The mechanism of internalization impacts intracellular trafficking, that determines how quickly a cargo is delivered to various organelles or degraded.

The driving force for the passage is the membrane potential. The process favors the idea that internalization is energy dependent and the ability of maintenance of membrane potential is dependent on ATP.

Fig. 11 Adaptive translocation mechanism of uptake

3. Tissue uptake:
Most of the studies determine the ability of transporters to enter the tissue is of critical importance, there is a major requirement for enhancing them towards therapeutic applications.

The guanidinium transporters shows a selective tissue uptake, thus they can be used to obtain selectivity by administering at local site of action. The tissue specific cleavage enhances the chances of release and translocation of active moiety at nearby tissue. It has wide scope for targeted delivery.
EXPERIMENTAL SECTION:

Infrared Spectra
Infrared spectra were recorded on shimadzu FTIR-8300 spectrometer. Spectra were calibrated against the polystyrene absorption at 1601 cm-1. Solid samples were recorded as KBr discs and liquids as thin films in between NaCl plates.
Nuclear magnetic resonance spectra:
1H NMR spectra were recorded on Bruker-Avance DPX-300 and DPX-500 instruments in CDCl3 solutions, unless otherwise stated. Chemical shifts are reported with respected to tetramethylsilane (Me4Si) as the internal standard (for 1H NMR) and the central line (77.16 ppm) of CDCl3 (for 13C NMR). The chemical shifts are expressed in parts per million (??) downfield from Me4Si. The standard abbreviations s, d, t, q and m refer to singlet, doublet, triplet, quartet, and multiplet respectively. Coupling constant (??), whenever discernible, has been reported in Hz.
Mass spectra:
High resolution mass spectra (HRMS) were measured in a QTOF I (quadrupole-hexapole-TOF) mass spectrometer with an original Z-spray-electrospray interface on Micro (YA-263) mass spectrometer (Manchester, UK).
Chromatography:
Reactions were monitored by thin layer chromatography (TLC). TLC was performed with silica gel 60 F254 aluminium sheets (Merck). Visualization of the spots on TLC plates was achieved either by using UV light or by charring with phosphomolybdic acid solution, ninhydrin solution, and potassium permanganate solution as required. Column chromatography was usually carried out with silica gel (230-400 mesh). The columns were usually eluted with petroleum ether-AcOEt mixtures. For polar compounds DCM-MeOH solvent systems were used. Petroleum ether refers to the fraction of boiling point 60-80oC.

General:
All reagents were purchased from commercial sources and used without further purification, unless otherwise mentioned. Solvents were purified and dried according to recommended Procedures. All moisture and sensitive reactions were performed under argon atmosphere with dry, freshly distilled solvents under anhydrous conditions using standard syringe-septum technique. Tetrahydrofuran (THF), benzene, toluene, diethyl ether, were distilled from sodium benzophenone ketyl. DCM, DMF, and DMSO were distilled freshly from sodium hydride. Amines were distilled over potassium hydroxide pellets and stored over the same.
A usual workup of the reaction mixture consists of extraction with common organic solvents (ether, DCM, AcOEt), washing with water, brine, drying over NaSO4, and then concentrated under reduced pressure on a rotary evaporator unless specified. Yields (isolated) were reported after purification of the crude either by column chromatography or by vacuum distillation.
For cell culture, reagents from Invitrogen were used. Cell culture dishes were obtained from BD Falcon. The fluorescent microscopic images were obtained in a Leica DM 3000 microscope. Luminescence was measured by integration over 10s in a Thermo Luminoskan Ascent using Promega Bright Glow assay kit.

1. Synthesis of 1-trityl piperidi-4-one:
To a solution of dry DMF Et3N and TrCl3 were added and stirred at RT for 12hr. The reaction mixture was concentrated under reduced pressure, redissolved in ethyl acetate washed with water and brine. The organic part was dried over NaSO4 and concentrated under reduced pressure the crude was purified by column chromatography (230-400 mesh) to get the desired product.(Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for


2. Synthesis of 1-tritylpiperidin-4-amine:
To a stirred solution of 3 in MeOH added ammonium biborate (NH4)2B4O7.4H2O and sodium borocyanide. The resulting mixture was heated at 65o c for 2hrs. The reaction mixture was concentrated under reduced pressure, redissolved in ethyl acetate and washed with water and brine. The organic part was dried over NaSO4 and concentrated under reduced pressure and purified by column chromatography to get the desired compound as white floppy compound. (Rf = , ). IR (neat/CHCl3); 1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

3. Synthesis of 1-benzoyl-3-(1-tritylpiperidin-4-yl)Thiourea:
To a stirred solution of 4 in DCM at 00 C temperature added benzyl isothiocyanate diluted with DCM dropwise. The reaction mixture was stirred at the same temperature for 1 hr. TLC shows complete consumption of starting material. Then concentrated under reduced pressure and crude was purified by column chromatography to get the desired product as pale yellow solid. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

4. Synthesis of ter-butyl piperazine-1-carboxylate:
To a stirred solution of 5 in DCM at 0o C temperature, (BOC)2O diluted with DCM added dropwise slowly over 15 min. The reaction mixture was stirred at 0oC to RT for 3 hr the mixture was concentrated under reduced pressure and then diluted with ethyl acetate and washed with water and brine. The organic part was dried over NaSO4 and concentrated under reduced pressure the crude was purified by column chromatography (230-400 mesh) to get the desired product.(Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

5. Synthesis of ter-butyl-4-(N`-benzoyl-N-(1-tritylpiperidin-4-yl)carbamimidoyl ) piperazine-1-carboxylate:
To a stirred solution of 4 and 6 in DMF at 0o c added DIPEA and HgCl2 diluted in DMF. Instantly it gives yellow colour precipitate and after 30 min it turns to black. The reaction mixture was stirred at 00 c for 1 hr. the precipitate was filtered through celite bed and the filtrate residue was purified by column chromatography to get the desired compound. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

6. Synthesis of (Z)-tert-butyl4-(N`-benzoyl-N-(piperidin-4-yl)carbmimidoyl) piperazine-1-carboxylate:
To a stirred solution of in DCM at 00c CF3CH2OH and then AcOH diluted with CF3CH2OH dropwise. After stirring at 0 C for 30 min. the reaction mixture was concentrated under reduced pressure and washed it with 5% EA-PE to get the desired product. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

To a stirred solution of A in DCM at 00 C temperature added phenyl isothiocyanate diluted with DCM dropwise. The reaction mixture was stirred at the same temperature for 1 hr. TLC shows complete consumption of starting material. Then concentrated under reduced pressure and crude was purified by column chromatography to get the desired product as pale yellow solid. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

To a stirred solution of A in EtOH added NH2??-NH'?2.H2??0 and heated at 90 c for 24 hrs. The organic part was dried over NaSO4 and concentrated under reduced pressure and purified by column chromatography to get the desired compound. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

RESULTS AND DISSCUSION

1. SYNTHETIC ATTEMPTS TOWARDS FLEXIBLE MOLECULAR TRANSPORTER:
As we already discussed the importance of molecular transporter and how guanidinium group facilitates cell transfection.
We had successfully developed the non-peptide based transporter and now synthetic attempts were made to enhance the steric hindrance of the transporter, thus it can be more flexible. The flexibility is the important pre requisite for a transporter so as to associates with the cell membrane to enhance the cellular translocation process.
There are many methods available for guanidylation, but the synthesis of hindered guanidine is a ever challenging task. Many synthetic attempts were made but doesn`t resulted a fruitful end.
Initial attempts:
Many methods were tried on trail and error basis to synthesize hindered guanidinium. But no method had given a better results.

2. CELL TRANSFECTION STUDIES:

The molecular transporter was tied to a fluorescent dye, BODIPY and analyzed for cell transfection.

3. APPLICATIONS TO CELLULAR DELIVERY:
Passage across the cell membrane is the main obstacle for the effective delivery of macromolecules in to cells; the transporter can serve to improve the efficiency of uptake of macromolecules in to cells invitro and invivo. The attachment of peptides and proteins to an agent that is able to cross membrane hence achieving cellular delivery is of growing importance.
i. Delivery of small particles:

The plasma membrane is impermeable not only to the large, polar bio-molecules but also small particles. Fluorescein is the small particle used to monitor the cell penetration ability as this small molecule cannot pass through barrier.

ii. Delivery of Peptides & Proteins:

As inspired from Tat, several peptides and proteins were delivered into the cytoplasm by conjugating with synthetic transporters, resulted in high accumulation in heart, liver, and spleen, low to moderate distribution in lungs and skeletal muscles. Hence it is assumed to have prophylactic and therapeutic potential.

It has shown the potential to target the oxidative stress process, so used in treating the disorders. When a transporter is tethered to peptide, it has shown the potential application in inflammatory conditions.

iii. Delivery of Nucleic Acids:

Introduction of genetic material in to the targeted cells to replace existing genetic material to accommodate functions that had been incapable of cell performance can be described as gene therapy.
Gene therapy using synthetic transporters holds a promising potential to treat various diseases, as conventional methods like viral and non-viral vectors suffers severe limitations in gene delivery.
As influenced by Tat peptide, our molecular transporter can efficiently uptake in to nucleus in time-, concentration-, dependent manner facilitated by cell surface proteoglycans.

iv. Delivery of Antibodies:

To achieve targeted tumoricidal effect, the antibodies are essentially delivered intracellularlly. Immunoglobulins cannot cross the membrane, however the conventional methods limits as it resulted in disruption of the membrane. The antibodies are fused with the synthetic transporter using recombinant fusion method described earlier in the text and delivered in to the targeted cells.
4. EXPERIMENTAL SECTION

1. Synthesis of ter-butyl piperazine-1-carboxylate:
To a stirred solution of 5 in DCM at 0o C temperature, (BOC)2O diluted with DCM added dropwise slowly over 15 min. The reaction mixture was stirred at 0oC to RT for 3 hr the mixture was concentrated under reduced pressure and then diluted with ethyl acetate and washed with water and brine. The organic part was dried over NaSO4 and concentrated under reduced pressure the crude was purified by column chromatography (230-400 mesh) to get the desired product.(Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of

To a stirred solution of A in acetonitrile added NaN3 and then heated at 700 c temperature for overnight. The RM was concentrated under reduced pressure redissolved in ethyl acetate acidified with HCl, extracted with ethyl acetate two times. The organic part was dried over NaSO4 and concentrated under reduced pressure and purified by column chromatography to get the desired compound as brown oil. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of

To a stirred solution of A in CH3CN added B and pyridine and stirred at RT for 3 hrs. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of

To a stirred solution of A in CH3CN added B (added twice) diluted with dry DCM followed by the addition of DIPEA. The reaction mixture was stirred at RT for overnight. Then concentrated under reduced pressure and crude was purified by column chromatography to get the desired product. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of
To a stirred solution of A in THF added DCM and TFA at 0o c. The RM reaction mixture was stirred at RT for 6 hrs. Then concentrated under reduced pressure and crude compound was used for the next step. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of
To a stirred solution of A in dry DCM added THF, DIPEA and B. the RM was stirred at RT for 12 hrs. Then concentrated under reduced pressure and crude was purified by column chromatography on silica gel (230-400 mesh) to get the desired product. (Rf = , ). IR (neat/CHCl3):;1H NMR (500 MHz, CDCl3):; 13C NMR(125 MHz, CDCl3):; HRMS (ESI) (M + H)+ calculated for

Synthesis of
To a stirred solution of A in EtOH added NH2??-NH'?2.H2??0 and heated at 90 c for 24 hrs. The organic part was dried over NaSO4 and concentrated under reduced pressure and purified by column chromatography to get the desired compound.

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Essay UK, Structural Components Of Plasma Membrane. Available from: <https://www.essay.uk.com/free-essays/science/structural-components-plasma-membrane.php> [21-05-19].


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