Protocol for synthesis of alpha 2,3-sialyllactose - (Jun/15/2007 )
Hi,
Does anyone have a protocol (preferably a simple one) for the synthesis of alpha 2,3-sialyllactose?
Thanks! 
Does anyone have a protocol (preferably a simple one) for the synthesis of alpha 2,3-sialyllactose?
Thanks!
if you do some google/pubmed search and tell us the link.. we will send u the protocol..
cheers
cheers
Hi Lab HelpDesk, (I'm in much need of your help
)Thanks for your reply.... I'm actually having a tough time finding information about alpha 2,3 sialyllactose (2,3 SL) synthesis. I've just come across this paper:
Lee et al. Enzyme and microbial technology, (2002), Vol 31, Pg 742
Which mentions something about the preparative synthesis of 2,3 SL using fetuin as a sialic acid donor. But I don't know if this is good enough to obtain a 100% pure sample of 2,3 SL. Maybe you'd have a better idea?
I also would like to synthesize alpha 2,6 sialyllactose but have no idea of how to go about it. I've seen this website http://www.nacalai.co.jp/en/reagent/Sialic_Acid.htm but don't know how it'll help.
Thanks once again for your help.... It's much appreciated. (I just hope someone has a protocol on synthesizing this stuff out there) Urgh.... Now I'm beginning to hate carbohydrate chemistry
don't be frusted sara.. everything should be ok..
anyways.. here is your aticle..
http://base.google.com/base_media?q=hand-7...3759&size=8
or http://base.google.com/base/a/2253887/D16739532304413210848
i am sorry i hv to post it here.. but i didn't hv ur email..
I'll reccomend you to try searching weily publications n protocols.. you might find some clue..
n keep googling.. i am sure u'll find somthing..
Oh god.... I'm really really lost now 
Can someone explain the following steps to me? I've never heard of half the methods (HPLC, ultrafiltration, sephadex, pool, etc)
So this paper says;
to add lactose and fetuin to phosphate buffer.
Then add 0.1 U of enzyme (is this cell free extract???) and conduct the reaction for 8 hours at RT.
Then monitor by HPLC (whatever that is).
Next, boil reaction mixture (to what temp for godsake!!) and centrifuge.
Protein removed by ultrafiltration (I'm confused again) with MW cut off: 10,000).
Filtrate applied to sephadex G-15 column with water as eluent.
Sialylated trisaccharide monitored by orcinal assay and unreacted lactose monitored by phenol/sulfuric acid assay.
Trisaccharide containing fractions pooled (what does this mean?), lyophilized and identified by HPLC and 300MHz proton NMR.
Does this method actually give me 2,3 SL as a product??? How how pure is the damn thing!! I'm just so irritated!! Why the hell can't things be kept simple in biochem?? This is driving me crazy.......
Can someone explain the following steps to me? I've never heard of half the methods (HPLC, ultrafiltration, sephadex, pool, etc)
So this paper says;
to add lactose and fetuin to phosphate buffer.
Then add 0.1 U of enzyme (is this cell free extract???) and conduct the reaction for 8 hours at RT.
Then monitor by HPLC (whatever that is).
Next, boil reaction mixture (to what temp for godsake!!) and centrifuge.
Protein removed by ultrafiltration (I'm confused again) with MW cut off: 10,000).
Filtrate applied to sephadex G-15 column with water as eluent.
Sialylated trisaccharide monitored by orcinal assay and unreacted lactose monitored by phenol/sulfuric acid assay.
Trisaccharide containing fractions pooled (what does this mean?), lyophilized and identified by HPLC and 300MHz proton NMR.
Does this method actually give me 2,3 SL as a product??? How how pure is the damn thing!! I'm just so irritated!! Why the hell can't things be kept simple in biochem?? This is driving me crazy.......
"Then add 0.1 U of enzyme (is this cell free extract???)"
-It says it is an enzyme.
"Next, boil reaction mixture (to what temp for godsake!!) and centrifuge."
-It will be 100 oC if you are not presently on the top of Mt. Himalaya . It is to kill the enzyme, I guess.
Protein removed by ultrafiltration (I'm confused again) with MW cut off: 10,000).
This filter removes all the molecules greater than 10,000 Da.
"Sialylated trisaccharide monitored by orcinal assay and unreacted lactose monitored by phenol/sulfuric acid assay.
Trisaccharide containing fractions pooled (what does this mean?), "
-Pool: You combine all the fractions that is orcinal reaction positive but phenol/H2SO4 negative.
"Does this method actually give me 2,3 SL as a product??? How how pure is the damn thing!! I'm just so irritated!! Why the hell can't things be kept simple in biochem?? This is driving me crazy......."
-HPLC analysis will tell you the purity.
This reaction is considered to be very simple. If you are making it through organic synthesis, it would take you a whole year to do. So, take it easy.
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Thanks.... Do you know how long it'll take to synthesize 3-SL? And is it ok to stop between steps (because I'm NOT working on this thing 24 hours a day) and store in the fridge? I'll try it over the next few weeks.... I just hope it works. If it doesn't then looks like i'll be posting some really harsh comments on this forum. I'm so stressed out..... 