Patent Application: US-52454705-A

Abstract:
it has been discovered that facile glycation of proteins can be achieved by colyophilization of a protein with a reducing sugar , subjecting the lyophilized mixture to a vacuum and incubating at an elevated temperature for 1 to 24 h . a stable ketoamine derivative is formed with amino groups in the protein and no advanced glycation end products are observed , as is the case with aqueous glycation procedures . another novel feature is that the in vacuo glycation reaction takes place with the protonated amine and not the deprotonated amine as is believed to be the case for aqueous glycation reactions . advantage can be taken of the in vacuo glycation reaction to achieve facile covalent cross - linking of proteins by lyophilizing protein or proteins with compounds containing two or more reducing sugars separated by a linker .

Description:
ribonuclease a ( sigma r - 4875 ) from bovine pancreas and human hemoglobin ( sigma h - 7379 ) were purchased from sigma chemical company and used without further purification . d -[ u - 14 c ] glucose ( 310 mci / mmol ), 50 μci in 250 μl aqueous solution containing 3 % ethanol was purchased from amersham pharmacia biotech . d - glucose - 1 - 13 c , 99 atom % 13 c , was purchased from sigma chemical company . all other chemicals , reagents and solvents used were high purity preparations obtained from commercial sources . typically , protein was added to a glucose solution for a final ratio of 5 : 1 or 10 : 1 protein / glucose ( w / w ). the ph of the solution was adjusted with 1m hcl or naoh , via micro - syringe , to the desired ph ( 6 . 5 to 10 ). aliquots of protein / reducing sugar solutions were transferred to borosilicate test tubes and frozen by immersing in liquid nitrogen . the samples were subsequently lyophilized to room temperature . the tubes , containing the lyophilized protein / reducing sugar mixture , were narrowed and sealed under vacuum ( 1 - 50 × 10 − 3 torr ) with an oxygen - enriched flame , placed in an oven at 65 ° c . and left to react for a controlled period of time ( 2 to 24 hours ). trace radio - glycation of rnase was prepared by dissolving rnase ( 20 mg , 1400 nmol ) in dh 2 o ( 10 ml ), adding d -[ u - 14 c ] glucose solution ( 20 μl , 13 nmol ; see above ), adjusting the ph with 1m naoh ( ph 6 . 5 or 10 ) and lyophilizing as described above . a hemoglobin / glucose solution ( 10 : 1 w / w ) was prepared containing d -[ u - 14 c ] glucose 37 . 5 nci per mg of protein . after application of the in vacuo reaction procedure , samples were dissolved in 10 % glucose solution to displace un - reacted d -[ u - 14 c ] glucose and extensively dialyzed against dh 2 o . aliquots of the protein / reducing sugar solution ( 50 μl ) were transferred to scintillation vials before and after dialysis , scintillation cocktail ( 5 ml ) ( aquasol - 2 , dupont ) was added and the samples were counted for 10 minutes on a beckman ls 6500 multi - purpose scintillation counter using the 14 c window . protein amount was normalized by recording absorbance at 280 nm on a pharmacia biotech ultraspec 2000 uv / v is spectrophotometer with dh 2 o as blank . rnase ( 106 . 54 mg ) was dissolved in 50 ml dh 2 o in a 250 ml rb flask , d - glucose - 1 - 13 c ( 10 . 60 mg ) was added to the protein solution , the ph was raised to 10 with 1 m naoh and the solution was frozen / shelled in n 2 ( l ) and lyophilized . the lyophilized protein : reducing sugar mixture was transferred to a borosilicate hydrolysis tube and glycated as above for 24 hours . rnase glycated with d - glucose - 1 - 13 c was extensively dialysed against dh 2 o ( mwco 3500 ), lyophilized and dissolved in 2 h 2 o ( 99 . 9 atom % 2 h , cambridge isotope laboratories ) and the 13 c - nmr spectrum was acquired on a bruker spectrometer operating at 9 . 4 tesla ( 13 c , 100 . 6 mhz ) for 600 scans using the dept 135 pulse sequence ( sanders and hunter , 1987 ). bifunctional cross - linkers containing reducing sugars separated by a spacer ( fig4 a ) were synthesized from glucose derivatives . glucosamine or glucuronic acid are glucose derivatives which contain functional groups through which the spacer can be coupled , a carboxylic group in the former and an amino group in the latter . bis acids and amines of vary lengths were used to furnish the linear spacers and were coupled to the appropriate glucose derivative using a water - soluble carbodiimide ( 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ). branched cross - linkers were synthesized from glucuronic acid and polypropyleneimine dendrimer ( generation 1 . 0 ) ( fig4 b ) using o -( 7 - azabenzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluroniumhexafluorophosphate ( hatu ) as coupling agent . typically , a stock solution of accurately weighed protein / ml of distilled h 2 o was prepared and , depending on the experiment , the ph was adjusted with either 1n naoh or 1n hcl . aliquots of the protein stock solution of a minimum volume of 500 μl to yield between 2 - 5 mg protein / tube , were transferred to 13 × 100 mm borosilicate glass disposable culture tubes to which varying aliquots of a solution of cross - linker dissolved in distilled h 2 o were added . the tubes were left to stand for approximately 10 - 15 minutes and were then flash frozen and lyophilized . the tubes were then sealed under vacuum using an oxygen - enriched flame and incubated at 60 ° c .- 85 ° c . for a period of 12 - 18 hours . the protein was reconstituted with 0 . 5 ml of distilled h 2 o and analyzed by sds - page , hplc or fplc . sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed using a bio - rad mini - protean ii dual slab cell apparatus . all reagents and molecular weight markers were purchased from biorad or sigma , and were electrophoresis grade quality ( buffers were prepared as required using dh 2 o ). approximately 10 μg of protein / well was loaded onto a 12 % polyacrylamide gel and electrophoresis was conducted at a constant current of 20 - 25 ma / gel ( with a voltage gradient of 70 - 150 mv ) under discontinuous gel conditions . coomassie brilliant blue g250 was used to stain the protein bands in the gel following electrophoresis . solution 13 c nmr of glycated rnase with 13 c 1 - d - glucose reveals that only one product is present . the single resonance peak , located at 53 . 2 ppm in fig1 , corresponds to the cyclic ketoamine adduct ( neglia , c . i ., cohen , h . j ., garber , a . r ., ellis , p . d ., thorpe , s . r . and baynes , j . w ., 1983 , j . biol . chem ., 258 , 14279 - 14283 , ed .). it is particularly notable that no advanced glycation end - products ( browning reaction ) are present as is the case in conventional non - reductive glycation procedures ( yaylayan , v . a . and huyghues - despointes , a ., 1994 , crit . rev . food sci . nutrition , 34 , 321 - 369 , ed .). conventional glycation procedures require the use of a reducing agent such as sodium borohydride to form a stable amino alcohol derivative and prevent the production of advanced glycation end - products . another advantage of the in vacuo glycation procedure is that no additional reaction is required to stabilize the glycation product as it yields a stable ketoamine product directly . d -[ u - 14 c ] glucose ( 310 mci / mmol ) was used directly as supplied without addition of any unlabeled glucose to determine the rate of glycation using the in vacuo procedure . fig2 shows that the rate of incorporation at ph 6 . 5 and ph 10 is very similar and is essentially complete after 12 hours with incubation at 65 ° c . fig3 shows that within experimental error all the amino groups of hemoglobin can effectively be glycated by the in vacuo glycation procedure . the accepted mechanism for the glycation in water involves the nuleophilic attack of a proteins amino group on the open chain form of the reducing sugar forming a schiff base which undergoes amadori rearrangement to form the ketoamine derivative . as lysine ε - amino groups in proteins normally have ionization constants of 10 . 5 to 11 , very little glycation of amino groups would be expected below ph 10 . 5 where the protonated form of the amino group predominates . however , the results in fig3 show that the protonated form of the amino group is readily glycated by the in vacuo procedure . the mechanism by which this glycation occurs has not been established but it is clearly different from that postulated for glycation in solution . the in vacuo glycation methodology has been used to develop a new class of protein cross - linking reagents , viz . linear bifunctional and multifunctional branched glyco - crosslinkers . two such cross - linking reagents have been synthesized , 4 , 7 , 10 - trioxa - 1 , 13 - tridecanediglucuronamide ( ttdg ) ( fig4 a ) and polypropyleneiminetetraglucuronamide dendrimer ( ptgd ) ( fig4 b ). fig5 shows cross - linking of human hemoglobin with increasing amounts of the bifunctional glyco - crosslinker ttdg using the in vacuo glycation methodology . substantial amounts of dimer and higher oligomers of cross - linked hemoglobin are clearly visible ( fig5 a ) with sds - page , as well some insoluble , very highly cross - linked material that does not enter the gel . fig5 b shows the effect of the addition of an excipient , trehalose which promotes the formation of soluble cross - linked oligomers . the glycation procedure with the branched glyco - crosslinker ptgd also yields substantial amounts of dimer and higher oligomers of hemoglobin ( fig6 ). the results obtained in the present study show that facile glycation of proteins can be achieved by an in vacuo reaction of the protein with a reducing sugar or compounds containing one or more reducing sugars . the in vacuo glycation procedure can be used with linear bifunctional and multifunctional branched glyco - derivatives containing reducing sugars to achieve facile covalent cross - linking of proteins . from the foregoing , it will be appreciated that , although specific embodiments of the invention have been described herein for purposes of illustration , various modifications may be made without deviating from the spirit and scope of the invention . 1 . sundaram , p . v . and r . venkatesh , retardation of thermal and urea induced inactivation of a - chymotrypsin by modification with carbohydrate polymers . protein eng ., 1998 . 11 ( 8 ): p . 699 - 705 . 2 . aoki , t ., et al ., improvement of heat stability and emulsifying activity of ovalbumin by conjugation with glucuronic acid through the maillard reaction . food res . int ., 1999 . 32 : p . 129 - 133 . 3 . mammen , m ., s .- k . choi , and g . whitesides , polyvalent interactions in biological systems : implications for design and use of multivalent ligands and inhibitors . angew . chem . int . ed ., 1998 . 37 : p . 2754 - 2794 . 4 . wong , s . y ., current opin . struct . biol ., 1995 . 5 : p . 559 - 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