Editing DTBP-polyAmine

== Materials  ==

Fluka: Spermine (Mw 202.35), dimethyl-3.3’-dithiobispropionate x 2 HCl DTBP; Mw 309.28) 

Sigma: Spermidine (Mw 145.25), pentaethylenhexamine (Mw 232.4), oligoethylenimine with an average molecular weight of 800 Da (OEI 800), 1,6-hexanediol-diacrylate (Mw 226.28) and branched PEI with an average molecular weight of 25 kDa (PEI25) 

Pierce: Dithiobis(succinimidylpropionate) (DSP; Mw 404.42) <br>Merck: Triethylentetramine (Mw 146.24) and tetraethylenpentamine (Mw 189.3) 

Dissolve 85 mg DTBP x 2 HCl (IP) in 1 ml of DMSO<br>Sonicate until it is completely dissolved (crosslinker stock). 

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== Synthesis  ==

[[Image:Polyamine structure.jpg|650px|Polyamine structure.jpg]] 

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=== Stability of Conjugated polymer  ===

DTBP--&gt; Imine, unstable<br> 

[[Image:Imine reaction.jpg]] 

DSP--&gt;Amide, more stable<br> 

[[Image:Amide hydrolysis 2.jpg]] 

[[Image:Amide hydrolysis.jpg]] 

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Hexanediol-Diacrylate--&gt;Ester, very unstable, hydrolysis rapidly<br> 

[[Image:Ester hydrolysis.jpeg|600px|Ester hydrolysis.jpeg]] 

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=== Synthesis  ===

[[Image:Polyamine reactions.jpg|650px|Polyamine reactions.jpg]] 



[[Image:Reduciable_linker.jpg]]



[[Image:Reduciable_linker_2.jpg]]

[[Image:Polyamine-reaction products.jpg|650px|Polyamine-reaction products.jpg]] 

[[Image:Polyamine hemolytic activity.jpg|650px|Polyamine hemolytic activity.jpg]] 

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[[Image:Polyamine transfection efficiency.jpg|650px|Polyamine transfection efficiency.jpg]] 

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=== [[Image:Linker ratio transfection efficiency.jpg]]  ===

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=== Polyamine-Hexanediol-Diacrylate (PA-HD)  ===

<br>Dissolve 300 mg 1.6-hexanediol-diacrylate (HD) in 1 ml DMSO (crosslinker stock)<br>Add 0.1 mmol polyamine to 0.05 mmol (PA-HD-0.5), 0.1 mmol (PA-<br>HD-1) or 0.2 mmol (PA-HD-2) crosslinker stock<br>incubate at 60 °C for 4 days under constant shaking (450 rpm, Eppendorf Thermomixer). 

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=== Polyamine-DTBP (PA-IP)  ===

[[Image:PEI DTBP synthesis.jpg]] 

Dissolve 85 mg DTBP x 2 HCl (IP) in 1 ml of DMSO<br>sonicate until it is completely dissolved (crosslinker stock).<br>Add 0.1 mmol polyamine to 0.05 mmol (PA-IP-0.5), 0.1 mmol (PA-IP-1) or 0.2 mmol (PA-IP-2) crosslinker stock 

incubate at 15 °C for 24 h under constant shaking (450 rpm, Eppendorf Thermomixer). 

OEI-IP-1 Mix 0.98 ml OEI 800 stock (400 mg/ml in DMSO) with 1.8 ml DTBP stock (83 mg/ml in DMSO) and reacted for 24 h at 15°C under constant shaking. 

'''Synthesis and purification by dialysis''':&nbsp;Yield is 76 mg lyophilized polymer (yield 17&nbsp;%). 

'''Purify polymer by SEC''': yeild is 42 mg lyophilized polymer (yield 43&nbsp;%). 

=== '''Scale up'''  ===

Small scale: Add 85.5 mg powder of DTBP x 2 HCl to 480 mg OEI 800 in 4 ml DMSO,react at 15 °C for 24 h under constant shaking. 

Medium scale: mix 0.98 ml OEI 800 stock (400 mg/ml in DMSO) with 1.8 ml DTBP stock (83 mg/ml in DMSO) 

React for 24 h at 15°C under constant shaking. 

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Purification by dialysis: &nbsp;yield is 76 mg lyophilized polymer (yield 17&nbsp;%). 

Purification by SEC: &nbsp;yield is 42 mg lyophilized polymer (yield 43&nbsp;%). <br> 

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=== Polyamine-DSP (PA-SP)  ===

Dissolve&nbsp;250 mg DSP (SP) &nbsp;in 1 ml of DMSO (crosslinker stock) 

Add 0.1 mmol&nbsp;polyamine to 0.05 mmol (PA-SP-0.5), 0.1 mmol (PA-SP-1) or 0.2 mmol&nbsp;(PA-SP-2) crosslinker stock 

incubate at 15 °C for 24 h under constant shaking&nbsp;(450 rpm, Eppendorf Thermomixer). 

=== Scale up  ===

Mix 2.48 ml OEI 800 stock (400 mg/ml in DMSO) with 1.0 ml DSP stock (250 mg/ml in DMSO), react for 24 h at 15°C under constant shaking. 

Synthesis and purification by dialysis: yield is 76 mg lyophilized polymer (yield 14&nbsp;%). 

Purify by SEC: yield is 100 mg lyophilized polymer (yield 54&nbsp;%). 

== <br>Purification  ==

Size exclusion chromatography (SEC) 

Dilute polymer samples 1:4 with ddH20 and adjust&nbsp;the pH &nbsp;to pH 6 (with HCl conc.) immediately 

Column (10/30) packed with Sephadex G25 superfine (molecular weight exclusion limit 5 kDa for globular proteins) using ddH2O as the eluent 

Load up to 2 ml of diluted reaction product (maximal 100 mg crude product in maximal 20&nbsp;% DMSO) onto the column. 

Flow rate of 1.0 ml/min Collect 1.0 ml fractions of were collected. Pool fractions containing the major amounts of polymer 

Freeze-dry. 

Dialysis: &nbsp;Dilute reaction product 1:4 with ddH2O adjust pH to pH 6 (with HCl conc.) immediately. Use via a Spectra/Por® membrane for 48 h against 4 l of ddH2O at 4°C. Change water every 24hrs. 

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== Stability  ==

Esters are hydrolyzed in aqueous solution at basic as well as at acidic pH and should be more stable at neutral pH. Amides are less susceptible towards hydrolysis but can be degraded via base-catalyzed hydrolytic cleavage. Therefore, pH should be adjusted to 6 (acidic) after reaction. Polymer should be stored in either DMSO/MeOH or dry form (freeze-dried).&nbsp;Amides undergo many chemical reactions, usually through an attack on the carbonyl breaking the carbonyl double bond and forming a tetrahedral intermediate. Thiols, hydroxyls and amines are all known to serve as nucleophiles. Owing to their resonance stabilization, amides are less reactive under physiological conditions than esters. They can be hydrolysed in hot alkali, as well as in strong acidic conditions. Acidic conditions yield the carboxylic acid and the ammonium ion while basic hydrolysis yield the carboxylate ion and ammonia. 

=== synthesis of amide  ===

[[Image:SimpleAmideFormationByCondensation.png|400px|SimpleAmideFormationByCondensation.png]] 

=== Hydrolysis  ===

'''Ester''' 

When in the presence of a nucleophile, an ester may undergo reaction leading to cleavage of the carbonyl carbon-ether bond as shown below 

[[Image:Hydrolysis of ester.jpg]] 

'''Amide''' 

Due to their electronic similarity to esters, amides can also undergo hydrolysis, but they are less susceptible to nucleophilic attack such as that involved in hydrolysis reactions, again due to resonance stabilization and the donating of nitrogen’s NBEs to the carbonyl carbon. This delocalization has the effect of reducing the electrophilicity (reduce the partial positive charge) of the carbonyl carbon, and thereby reducing its ability to undergo attack by nucleophiles such as water or hydroxide in hydrolysis reactions: 

[[Image:Hydrolysis of amides.jpg]] 

Oxygen is more electronegative than nitrogen, thus it is less&nbsp;likely to donate its NBEs to the adjacent carbonyl. As a result, there is a lower degree of resonance delocalization through the ester carbonyl than there is in the amide carbonyl. This results in ester carbonyl carbons being relatively more electron deficient, or more electrophilic, than amide carbonyl carbons. This difference in carbonyl electrophilicity accounts for the difference in reactivity between esters and amides. It is important to understand that amides do undergo hydrolysis! The difference is the "ease" or rate at which hydrolysis occurs. Amides undergo hydrolysis at a slower rate than structurally related esters under comparable reaction conditions. 

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It is important to understand that amides do undergo hydrolysis! The difference is the "ease" or rate at which hydrolysis occurs. Amides undergo hydrolysis at a slower rate than structurally related esters under comparable reaction conditions. Consider the general example below. When placed in an aqueous alcohol solvent in the presence of base, the hydrolysis of the ester shown below may be&nbsp;complete in several hours. Under the same reaction conditions, it may take days (or longer) for the structurally similar amide to hydrolyzed! 

[[Image:Amide ester hydrolysis.jpg]] 

[[Category:Transfection]] [[Category:Paul]]