Enzymic synthesis of N-acetyl-D-mannosamine.

As repor ted earlier 1, an e n z y m e purif ied f rom Clostridium per#ingens (N-ace ty lneuramin ic acid aldolase; NaNaldolase) ca ta lyzes t he following reversible reac t ion: p y r u v a t e + N-acetyl-Dm a n n o s a m i n e (N-AcMm) ~ N-ace t y l neu rami n i c acid (sialic acid). The recogni t ion of N A c M m as a na tu ra l l y occurr ing suga r leads to t h e ques t ion of t he m e c h a n i s m of b iosyn thes i s of th i s compound . As ind ica ted below, N A c M m is fo rmed f rom ur id ine d i p h o s p h a t e ace ty lg lucosamine (UDPAG) b y ex t r ac t s ob ta ined f rom ra t liver. A recen t repor t ~ ind ica ted t h a t U D P A G is conver t ed to N-ace ty lga l ac tosamine (N-AcGalm) b y ra t l iver ex t rac t s . I n t he p re sen t s tudies , a t t e m p t s to repea t these e x p e r i m e n t s unde r t he s a m e condi t ions yie lded N-AcMm, b u t no de tec tab le N-AcGalm. I n c u b a t i o n m i x t u r e s were prepared as descr ibed ~ us ing commerc ia l U D P A G * * ; these were deprote inized wi th Ba(OH)2 and ZnSO 4, and deionized wi th mixed-bed , ion -exchange resin. The presence of N A c M m in t he so lu t ion was d e m o n s t r a t e d as follows: (I) Pape r c h r o m a t o g r a p h y on bora ted pape r 1,3 yielded a single N-acylh e x o s a m i n e spo t which mi g ra t ed a t 0. 4 t i m e s t he ra te ob ta ined wi th N-ace ty l -D-g lucosamine (N-AcGm). Th i s va lue for RN.AcGm cor responds to t h a t ob ta ined wi th e i ther N A c M m or NAcGa lm since these suga r s do no t sepa ra te unde r these condi t ions . (2) The add i t ion of p y r u v a t e a n d NaNaldo lase to t he u n k n o w n N a c y l h e x o s a m i n e yielded N-ace ty lneu ramin i c acid to t h e s a m e e x t e n t as t h a t ob ta ined wi th an equ iva l en t a m o u n t of N-AcMm. Nei ther N A c G m nor N-AcGa lm is ac t ive in t he NaNaldo lase s y s t e m 1. (3) Acid hydro lys i s of t he u n k n o w n N a c y l h e x o s a m i n e gave a h e x o s a m i n e which upon t r e a t m e n t wi th n i n h y d r i n a t p H 5.04 yielded a rab inose 5. Lyxose was no t detected. Bo th g lucosamine and m a n n o s a m i n e yield a rab inose unde r these condi t ions , whereas ga l ac tosamine yields lyxose. (4) C h r o m a t o g r a p h y of t he u n k n o w n h e x o s a m i n e on ion -exchange resin 1,~ resul ted in a single h e x o s a m i n e peak wh ich cor responded in pos i t ion to t h a t ob ta ined wi th D-mannosamine , b u t differed f rom t h a t ob ta ined wi th D-galac tosamine . There was no de tec tab le convers ion of N A c G m to N A c M m by t he ra t l iver ex t rac t s . For isolat ion and more comple te cha rac te r i za t ion of t he h e x o s a m i n e fo rmed f rom U D P A G , t he following large-scale i ncuba t ion m i x t u r e was p repa red : crude ra t l iver ex t r ac t (2z ml), 400 pmoles U D P A G , 1.75 m m o l e s MgSO4, 1.75 m m o l e s cysteine, 5 mmole s Tris . -HCl buffer (pH 7.5), to ta l vo lume 42 ml. After 2 h a t 37 °, 228 p m o l e s N a c e t y l h e x o s a m i n e were fo rmed as de t e rmined 7 by a modif ied Morgan-E l son reac t ion wi th syn the t i c N A c M m as s t anda rd . Fol lowing t h e add i t i on of o.2o M ZnSO, and o.zo M B a ( O H ) , (12o ml each), f i l trat ion, de ionizat ion of t he m i x t u r e wi th mixed -bed resin, and dialysis, t h e d ia lysa te was concen t ra ted , in vacuo. The concen t ra t e was hyd ro lyzed wi th HC1, t he resu l t ing h e x o s a m i n e isolated by ion-exchange ch roma tography1 , *, a n d crysta l l ized as t he hydroch lor ide f rom m e t h a n o l (first crop, 2o mg). After recrysta l l izat ion, t he ma te r i a l exh ib i ted t he following ana lyses :