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       ÀÌÇö¼ö ±³¼ö        2016.01.07 16:28        12691
´Ù¿î·Îµå : È¿¼Ò¸¦ ÀÌ¿ëÇÑ Ãµ¿¬¹° À¯µµÃ¼ÀÇ ±Û¸®ÄÚ½ÇÈ­_ÀÌÇö¼ö ±³¼ö.pdf(460 Kb)
 

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                                                                                                                            ¼­°­´ëÇб³ È­Çаú ÀÌÇö¼ö ±³¼ö

 

1. ¼­·Ð
ź¼öÈ­¹°(carbohydrates)Àº ¸¹Àº »ýü ³»ÀÇ ºÐÀڵ鿡¼­ ¹ß°ßµÇ°í, ´Ù¾çÇÑ »ý¸í Çö»ó¿¡¼­ Áß¿äÇÑ ¿ªÇÒÀ» ÇÑ´Ù. õ¿¬¹° ¶Ç´Â õ¿¬¹° À¯µµÃ¼·ÎºÎÅÍ °³¹ßµÈ »ý¸®È°¼ºÀ» °¡Áø ºÐÀÚµéÀº ź¼öÈ­¹°À» Æ÷ÇÔÇÏ°í ÀÖ´Â °æ¿ì°¡ ¸¹´Ù. ÀÌ·¯ÇÑ »ý¸®È°¼º ºÐÀڵ鿡¼­ ¹ß°ßµÇ´Â ź¼öÈ­¹°Àº ±× ¾à¹°ÀÇ È¿´É ¹× ¾à¹°µ¿¿ªÇÐ (pharmacokinetics) µî¿¡ °áÁ¤ÀûÀÎ ¿ªÇÒÀ» ÇÑ´Ù. ¿¹¸¦ µé¾î, Àß ¾Ë·ÁÁø Ç×»ýÁ¦ ÁßÀÇ ÇϳªÀÎ vancomycin¿¡ Á¸ÀçÇÏ´Â ÀÌ´çü (disaccharide)´Â ÀÌ ¾à¹°ÀÇ È¿´É¿¡ ¸Å¿ì Áß¿äÇؼ­, ÀÌ ÀÌ´çü¸¦ Á¦¿ÜÇÑ vancomycinÀº °ÅÀÇ ¾àÈ¿¸¦ ³ªÅ¸³»Áö ¾Ê°í, ¶Ç ÀÌ·¯ÇÑ Åº¼öÈ­¹°ÀÇ º¯ÇüÀº ÀüÇô ´Ù¸¥ ÇüÅÂÀÇ ¾àÈ¿¸¦ º¸À̱⵵ ÇÑ´Ù. ¶Ç ´Ù¸¥ õ¿¬¹°ÀÎ digitoxinÀº »ï´çü¸¦ Æ÷ÇÔÇÏ°í ÀÖ°í, ÀÌ »ï´çü¸¦ ´Ù¾çÇÑ ´Ü´çü·Î ġȯÇϸé, º¸´Ù Çâ»óµÈ Ç×¾ÏÈ¿°ú¿Í µ¶¼ºÀÇ °¨¼Ò¸¦ ³ªÅ¸³»¾ú´Ù.(±×¸² 1) ÀÌ·¯ÇÑ Á߿伺 ¶§¹®¿¡ õ¿¬¹°¿¡ Á¸ÀçÇϴ ź¼öÈ­¹°À» º¯ÇüÇÏ¿© ½Å¾àÀ» °³¹ßÇÏ·Á´Â ½Ãµµ°¡ ²ÙÁØÈ÷ ÁøÇàµÇ°í ÀÖ´Ù. ±×·¯³ª, õ¿¬¹°ÀÇ Åº¼öÈ­¹°À» º¯ÇüÇÒ ¼ö ÀÖ´Â È­ÇÐÀû ¹æ¹ýÀÇ ºÎÀç·Î ÀÎÇؼ­ °ý¸ñÇÒ ¸¸ÇÑ ÁøÀüÀÌ ¾ø´Â »óÅÂÀÌ´Ù. º» ÃѼ³¿¡¼­´Â È¿¼Ò, ƯÈ÷ ±Û¸®ÄÚ½ÇÆ®·£½ºÆÛ¶óÁ¦ (glycosyltransferases, GTs)¸¦ ÀÌ¿ëÇÑ ¾à¹° ¶Ç´Â ÀúºÐÀÚ È­ÇÕ¹°ÀÇ ±Û¸®ÄÚ½ÇÈ­, ƯÈ÷ ´Ù¾çÇÑ ±¸Á¶ÀÇ ´çÀ» µµÀÔÇÏ´Â glycorandomization¿¡ ´ëÇÑ ³»¿ëÀ» ¼Ò°³ÇÏ°íÀÚ ÇÑ´Ù.

 

                                      

                                            

                                                 ±×¸²1 . Vancomycin°ú digitoxinÀÇ ±¸Á¶.


2. ±Û¸®ÄÚ½ÇÈ­¿¡ Áß¿äÇÑ È¿¼Òµé
»ý¹°Ã¼ ³»¿¡ Á¸ÀçÇϴ ź¼öÈ­¹°À» Æ÷ÇÔÇÑ ºÐÀÚµéÀº ´ëºÎºÐ È¿¼Ò¿¡ ÀÇÇÑ ±Û¸®ÄÚ½ÇÈ­¿¡ ÀÇÇؼ­ ÇÕ¼ºµÈ´Ù. ¿©±â¼­´Â glycorandomization¿¡ ÀÌ¿ëµÉ ¼ö ÀÖ´Â È¿¼Òµé¿¡ ´ëÇؼ­ »ìÆ캸°íÀÚ ÇÑ´Ù (±×¸² 2). ÀÚ¿¬¿¡¼­ ±Û¸®ÄÚ½ÇÈ­¸¦ À§ÇØ »ç¿ëµÇ´Â ¸¹Àº È¿¼ÒµéÀÌ ÀÖÁö¸¸, glycorandomization¿¡ ÀÌ¿ëµÉ ¼ö ÀÖ´Â ÀûÀýÇÑ È¿¼Ò¸¦ ¼±ÅÃÇϴµ¥ À־´Â ´Ù¾çÇÑ ±âÁú¿¡ ÀÛ¿ëÇÒ ¼ö ÀÖ´Â ³ÐÀº ±âÁú ¼±Åüº (broad substrate specificity)°ú ´Ù¾çÇÑ ±âÁú¿¡ ´ëÇÑ È¿¼ÒÀÇ È¿À²¼º, ±×¸®°í È¿¼Ò¿Í ±âÁúÀÇ ÇÕ¼ºÀÇ ÆíÀǼº (availability)ÀÌ °í·ÁµÇ¾î¾ß ÇÑ´Ù.

 

2-1. ±Û¸®Äڽõ¥ÀÌÁî (glycosidases)¿Í Æ®·£½º±×¸®ÄÚ½ÇÈ­ (transglycosylation)
±Û¸®Äڽõ¥ÀÌÁî´Â ÀûÀýÇÑ leaving groupÀ» °¡Áö´Â ÀÌ´ç, »ï´ç, ¿Ã¸®°í´ç ¶Ç´Â ±Û¸®ÄÚ»çÀ̵å (glycosides)·ÎºÎÅÍ ´çÀ» ¹Þ¾ÆµéÀÌ´Â ºÐÀÚ (sugar-acceptors)¿¡ ´çÀ» Àü´ÞÇÏ´Â ¹ÝÀÀ¿¡ ÀÛ¿ëÇÏ´Â È¿¼ÒÀÌ´Ù. ÀÌ·¯ÇÑ ±Û¸®ÄÚ½ÇÈ­´Â ³ôÀº À§Ä¡/ÀÔü ¼±ÅüºÀ» °¡ÁöÁö¸¸, ¿­¿ªÇÐÀû º¯¼ö¿Í °¡¼öºÐÇØ ¹ÝÀÀ°úÀÇ °æÀï µî¿¡ ÀÇÇؼ­ È¿À²ÀÌ ¶³¾îÁö´Â ÇѰ踦 °¡Áø´Ù. ±×¸®°í ¿øÇü (wild type) ±Û¸®Äڽõ¥ÀÌÁî´Â ¾à¹° À¯»çü³ª õ¿¬¹°À» ±âÁú·Î ¹Þ¾ÆµéÀÌÁö ¾Ê±â ¶§¹®¿¡ º¯ÇüµÈ ±Û¸®Äڽõ¥ÀÌÁî°¡ ±Û¸®ÄÚ½ÇÈ­¿¡ ÀÌ¿ëµÇ¾î ¿Ô´Ù. ÀÌ·¯ÇÑ º¯Çü ±Û¸®Äڽõ¥ÀÌÁî¿¡´Â ±Û¸®ÄÚ½ÅÅ×ÀÌÁî (glycosynthases), Ƽ¿À±Û¸®ÄÚ¶óÀÌ°ÔÀÌÁî (thioglycoligases), Ƽ¿À±Û¸®ÄÚ½ÅÅ×ÀÌÁî(thioglycosynthases), O-±Û¸®ÄÚ¶óÀÌ°ÔÀÌÁî(O-glycoligases) µîÀÌ ÀÖ´Ù. ÀÌ º¯ÇüµÈ È¿¼Òµé¿¡¼­ ³ªÅ¸³ª´Â °øÅëÁ¡Àº ¿øÇü È¿¼Ò È°¼ººÎÀ§¿¡ Á¸ÀçÇϴ ģÇÙü (´ëü·Î ¾Æ½ºÆÄƽ »êÀ̳ª ±Û·çŸ¹Î »ê)°¡ ´Ù¸¥ ºñÈ°¼º ¾Æ¹Ì³ë»êÀ¸·Î º¯ÇüµÇ¾ú°í, ±Û¸®ÄÚ½ÇÈ­ È¿À²ÀÇ Çâ»óÀ» À§Çؼ­ ´Ù¾çÇÑ È°¼ºÈ­µÈ ±Û¸®ÄÚ½Ç Àü´Þü (glycosyl halides³ª aromatic glycosides)¸¦ »ç¿ëÇß´Ù´Â Á¡ÀÌ´Ù.


2-2. ±Û¸®Ä­Æ÷½ºÆ÷¸±·¹ÀÌÁî (glycan phosphorylases)
±Û¸®Ä­Æ÷½ºÆ÷¸±·¹ÀÌÁî´Â ´çÀÇ phosphorylysis¸¦ ÅëÇؼ­ sugar-1-phosphatesÀÇ Çü¼ºÀ» Ã˸ÅÇÑ´Ù. ±Û¸®Äڽõ¥ÀÌÁî¿¡ ÀÇÇÑ ±Û¸®ÄÚ½ÇÈ­¿Í ºñ±³Çؼ­ ±Û¸®Ä­Æ÷½ºÆ÷¸±·¹ÀÌÁîÀÇ ÀåÁ¡Àº °¡¼öºÐÇØ¿Í °°Àº °æÀïÇÏ´Â ºÎ¹ÝÀÀÀÌ °ÅÀÇ ¾ø´Ù´Â °ÍÀÌ´Ù. ÃÖ±Ù¿¡ ÀÌ È¿¼Ò¸¦ ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ ±Û¸®ÄÚ½Ç ¼ö¿ëü (acceptor)¿¡ ±Û¸®ÄÚ½ÇÈ­¸¦ ¼º°øÇÑ ¿¹°¡ ¸î °¡Áö ¹ßÇ¥µÇ¾ú´Ù. ¾ÕÀ¸·Î ÀÌ È¿¼ÒÀÇ º¯ÇüÀ» ÅëÇؼ­ ÀÌ È¿¼Ò¸¦ ÀÌ¿ëÇÏ¿© ±Û¸®ÄÚ½ÇÈ­¸¦ ½Ãų ¼ö ÀÖ´Â ±âÁúÀÌ ´Ù¾çÈ­µÉ °ÍÀ¸·Î ±â´ë°¡ µÈ´Ù. ±×·¯³ª glycorandomizationÀ¸·ÎÀÇ ÀÀ¿ë¼ºÀÇ È®´ë¸¦ À§Çؼ­´Â ´Ù¾çÇÑ sugar-1- phosphate¸¦ ½±°Ô ¾òÀ» ¼ö ÀÖ´Â ¹æ¹ýÀÇ °³¹ßÀÌ ¼ö¹ÝµÇ¾î¾ß ÇÑ´Ù.


2-3. ±Û¸®ÄÚ½ÇÆ®·£½ºÆÛ·¹ÀÌÁî (Glycosyltransferases, GTs)
À§¿¡¼­ ¾ð±ÞÇÑ È¿¼Ò¿Í ºñ±³ÇÒ ¶§, GTs´Â ´Ù¾çÇÑ ±âÁú¿¡ ´ëÇؼ­ ±Û¸®ÄÚ½ÇÈ­¸¦ ÁøÇà½ÃÅ°´Â °ÍÀ¸·Î ¾Ë·ÁÁ® ÀÖ°í, 65,000°³ (´ëºÎºÐÀÌ Leloir-type GTs, Áï ±Û¸®ÄÚ½Ç Àü´Þü·Î sugar nucleotides¸¦ »ç¿ëÇÏ´Â GTsÀÌ´Ù)°¡ ³Ñ´Â GTs°¡ ¾Ë·ÁÁ® Àֱ⠶§¹®¿¡ GTs´Â glycorandomizationÀ» À§Çؼ­ º¸´Ù È¿°úÀûÀÎ È¿¼Ò¶ó°í º¼ ¼ö ÀÖ´Ù. °Ô´Ù°¡ GT º¯Çüü ¶óÀ̺귯¸®¸¦ HTS (high-throughput screening) ÇÒ ¼ö ÀÖ´Â ¹æ¹ýµéÀÌ °³¹ßµÇ¾î¼­ Á» ´õ °³·®µÈ GTs¸¦ ¾òÀ» ¼ö ÀÖ´Â °¡´É¼ºÀÌ ³ô´Ù. ÇÑ°¡Áö ´ÜÁ¡Àº GTsÀÇ ±Û¸®ÄÚ½Ç Àü´Þü·Î »ç¿ëµÇ´Â sugar nucleotidesµéÀÌ ½ÃÁß¿¡¼­ ±¸ÀÔÇÒ ¼ö ÀÖ´Â °ÍÀÌ ¸¹Áö ¾Ê´Ù´Â Á¡ÀÌ´Ù. ±×·¯³ª ÃÖ±Ù ÀÌ·¯ÇÑ ÇѰ踦 ±Øº¹ÇÒ ¼ö ÀÖ´Â ¹æ¹ýµéÀÌ °³¹ßµÇ°í Àֱ⠶§¹®¿¡, GTs¸¦ ÀÌ¿ëÇÑ Á» ´õ È®ÀåµÈ glycorandomization °ü·Ã ¿¬±¸µéÀÌ °è¼Ó ÁøÇàµÉ °ÍÀ¸·Î ±â´ëµÈ´Ù.


3. È¿¼Ò¸¦ ÀÌ¿ëÇÑ sugar nucleotides ÇÕ¼º

 

              

 

               

                            ±×¸²2 . È¿¼Ò¸¦ ÀÌ¿ëÇÏ¿© Glycorandomized library¸¦ ±¸ÃàÇϴµ¥ »ç¿ëµÇ´Â ÀϹÝÀûÀÎ ¹æ¹ýµé.

 

È¿¼Ò¸¦ ÀÌ¿ëÇÑ glycorandomizationÀÌ È¿À² ¹× À§Ä¡/ÀÔü ¼±Åüº ¸é¿¡¼­ ÀåÁ¡ÀÌ ÀÖÁö¸¸, °¡Àå Å« °É¸²µ¹·Î ÀÛ¿ëÇÏ´Â °ÍÀÌ sugar donors¸¦ ¾ò±â°¡ Èûµé´Ù´Â Á¡ÀÌ´Ù. ƯÈ÷ GT¸¦ ÀÌ¿ëÇÑ glycorandomization¿¡¼­´Â sugar nucleotide°¡ donor·Î »ç¿ëµÇ°í, ¾Õ¿¡¼­µµ ¾ð±ÞÇßµíÀÌ, ´Ù¾çÇÑ sugar nucleotide¸¦ ¾ò´Â °ÍÀÌ ¸Å¿ì ¾î·Æ´Ù. °¡Àå Á÷Á¢ÀûÀÎ ¹æ¹ýÀº ÀÌ·¯ÇÑ donor¸¦ È­ÇÐÀûÀ¸·Î ÇÕ¼ºÇÏ´Â °ÍÀÌÁö¸¸, ´Ù¾çÇÑ ±¸Á¶ÀÇ ´ç¿¡ ´ëÇؼ­ sugar nucleotide library¸¦ ¸¸µå´Â µ¥´Â ź¼öÈ­¹°ÀÇ È­ÇÐÀû Ư¼ºÀ» °í·ÁÇÒ ¶§, ±Ùº»ÀûÀÎ ÇÑ°è°¡ ÀÖ´Ù. µû¶ó¼­ ¿©±â¼­´Â ÃÖ±Ù¿¡ ¿¬±¸µÈ È¿¼Ò¸¦ ÀÌ¿ëÇÑ sugar nucleotideÀÇ ÇÕ¼º¿¡ °üÇÏ¿© »ìÆ캸°íÀÚ ÇÑ´Ù. (±×¸² 3)


3-1. NDP-sugar »ýÇÕ¼º °úÁ¤À» °³·®ÇÑ ´ÙÁß È¿¼Ò¿¡ ÀÇÇÑ ÇÕ¼º
ÀÌ ¹æ¹ýÀº in vivo¿Í in vitro¿¡¼­ ÁøÇàµÇ´Â °úÁ¤À¸·Î ³ª´­ ¼ö ÀÖ´Ù. in vivo¿¡¼­ »ç¿ëÇÏ´Â ¹æ¹ýÀº ÀÚ¿¬¿¡ Á¸ÀçÇÏ´Â NDP-sugarÀÇ »ýÇÕ¼º °úÁ¤¿¡ °ü¿©ÇÏ´Â È¿¼ÒµéÀ» 2Â÷ ´ë»ç¹°À» »ý»êÇÏ´Â ¹Ì»ý¹°¿¡¼­ ¹ßÇö½ÃÄѼ­ ´Ù¾çÇÏ°Ô ±Û¸®ÄÚ½ÇÈ­µÈ 2Â÷ ´ë»ç¹°À» ¾òÀ» ¼ö ÀÖ´Ù. in vitro¿¡¼­ »ç¿ëÇÏ´Â ¹æ¹ýÀº ¿ì¼± NDP-sugarÀÇ »ýÇÕ¼º °úÁ¤¿¡ °ü¿©ÇÏ´Â È¿¼ÒµéÀ» ¹ßÇöÇÏ°í, Á¤Á¦ÇÏ¿© NDP-sugar¸¦ ÇÕ¼ºÇÏ´Â ¹æ¹ýÀÌ´Ù. ÀÌ ¹æ¹ýµéÀ» ÀÌ¿ëÇÏ¿© NDP-sugar¸¦ ÇÕ¼ºÇÏ´Â °ÍÀº °¡´ÉÇÏÁö¸¸, ¿øÇü È¿¼ÒÀÇ È°¼º¿¡ ÀÇÁ¸Çϱ⠶§¹®¿¡ ´Ù¾çÇÑ NDP-sugar¸¦ ¾ò´Â µ¥´Â ÇÑ°è°¡ ÀÖ´Ù.

 

                                 

            ±×¸²3 . GlycorandomizationÀ» À§ÇØ »ç¿ëµÇ´Â NDP-sugar libraryÀÇ ÇÕ¼ºÀ» À§ÇÑ ÀϹÝÀûÀÎ ¹æ¹ý.


3-2. ÀÌÁß È¿¼Ò¿¡ ÀÇÇÑ ÇÕ¼º
ÀÚ¿¬¿¡¼­ ãÀ» ¼ö ÀÖ´Â °¡Àå ªÀº NDP-sugar ÇÕ¼º ·çÆ®´Â Ä«À̳ªÁ¦(kinase)°¡ ¾Æ³ë¸Ó ÀλêÈ­ (anomeric phosphorylation)¿¡ ÀÇÇØ sugar-1-phosphate¸¦ ÇÕ¼ºÇÏ°í, ±× ´ÙÀ½ ´ºÅ¬·¹¿ÀµðµôÆ®·»½ºÆä¶óÁ¦ (nucleotidyltransferases, NTs)°¡ ÀÌ¹Ì ÇÕ¼ºµÈ sugar-1-phosphate¿¡ ´ºÅ¬·¹¿ÀƼµå¸¦ ´õÇÏ´Â °úÁ¤ÀÌ´Ù (±×¸² 3). ÀÌ ÇÕ¼º ·çÆ®¸¦ ÀÌ¿ëÇÏ¸é ºñ±³Àû ½±°Ô ´Ù¾çÇÑ NDP-sugar¸¦ ÇÕ¼ºÇÒ ¼ö ÀÖ´Ù.
´Ù¾çÇÑ sugar-1-phosphateÀÇ ÇÕ¼º¿¡´Â ÁÖ·Î ¾Æ³ë¸Ó Ä«À̳ªÁ¦ (anomeric kinase)°¡ »ç¿ëµÈ´Ù. ¾Æ³ë¸Ó Ä«À̳ªÁ¦´Â NTPÀÇ ¥ã-phosphate°¡ sugarÀÇ ¾Æ³ë¸Ó À§Ä¡·Î µµÀԵǴ ¹ÝÀÀÀ» Ã˸ÅÇÏ¿© sugar-1-phosphate¸¦ ÇÕ¼ºÇÑ´Ù. ÇöÀç±îÁö ¾ÆÁÖ ´Ù¾çÇÑ ¾Æ³ë¸Ó Ä«À̳ªÁ¦°¡ sugar-1- phosphate¸¦ ÇÕ¼ºÇϴµ¥ »ç¿ëµÇ¾ú´Ù. °ø°£ÀÇ ºÎÁ·À¸·Î ¿©±â¼­´Â ÀÚ¼¼È÷ ¾ð±ÞÇÒ ¼ö ¾øÁö¸¸ °¢ Ä«À̳ªÁ¦°¡ ¾î¶² sugar¸¦ ±âÁú·Î »ç¿ëÇÒ ¼ö ÀÖ´ÂÁö´Â Âü°í¹®Çå¿¡ Àß Á¤¸®µÇ¾î ÀÖ´Ù.

 

                        

                      

±×¸²4 . 2-chloro-4-nitrophenyl glycoside¸¦ ÀÌ¿ëÇÑ NDP-sugar library ÇÕ¼ºÀ» ÀÀ¿ëÇÏ¿© ±Û¸®ÄÚ½ÇÈ­¸¦ °¡´ÉÇÏ°Ô ÇÏ´Â GT¸¦ ã±â À§ÇÑ high- throughput, colorimetric screening ¹æ¹ý.


NTs´Â sugar-1-phosphate¿Í NTPs·ÎºÎÅÍ NDP-sugar¸¦ ÇÕ¼ºÇÏ´Â ¹ÝÀÀÀ» °¡¿ªÀûÀ¸·Î Ã˸ÅÇÑ´Ù. ¾Æ³ë¸Ó Ä«À̳ªÁ¦¿Í ÇÔ²² NTsµµ ´Ù¾çÇÑ Á¾·ùÀÇ È¿¼Ò°¡ ¿¬±¸µÇ¾î ºñ±³Àû ±¤¹üÀ§ÇÑ sugar-1-phosphate¿¡ ÀÛ¿ëÇÏ¿© NDP-sugar¸¦ ÇÕ¼ºÇÏ´Â NTs°¡ º¸°íµÇ¾ú´Ù. ÀÚ¼¼ÇÑ NTsÀÇ Á¾·ù¿Í °¢ NT¿¡ ±âÁú·Î »ç¿ëµÇ´Â sugar-1-phosphates´Â ¿ª½Ã Âü°í¹®Çå¿¡ Àß Á¤¸®µÇ¾î ÀÖ´Ù. ÇöÀç±îÁö ¾Æ³ë¸Ó Ä«À̳ªÁ¦¿Í NTs¸¦ ÀÌ¿ëÇÑ ÀÌÁß È¿¼Ò ¹æ¹ý¿¡ ÀÇÇØ 180¿©°³ÀÇ NDP-sugars°¡ ÇÕ¼ºµÇ¾ú°í, ÀÌ Áß¿¡´Â ´ÙÀ½ °úÁ¤¿¡¼­ labeling µî¿¡ ÀÌ¿ëµÉ ¼ö ÀÖ´Â Èï¹Ì·Î¿î ÀÛ¿ë±â (¿¹¸¦ µé¾î azide °°Àº ÀÛ¿ë±â)¸¦ °¡Áö°í ÀÖ´Â °Íµéµµ Æ÷ÇԵǾî ÀÖ´Ù.

 

                                                                     Ç¥ 1. ´ëÇ¥ÀûÀÎ glycorandomized libraries.

                     


 

3-3. GTÀÇ °¡¿ª¼ºÀ» ÀÌ¿ëÇÑ ¹æ¹ý
GT¿¡ ÀÇÇÑ ±Û¸®ÄÚ½ÇÈ­ ¹ÝÀÀÀº °¡¿ªÀûÀ̱⠶§¹®¿¡ ÀÌ ¹ÝÀÀÀÇ ÆòÇüÀ» ¿ª¹ÝÀÀ ÂÊÀ¸·Î Ä¡¿ìÄ¡°Ô Çϸé NDP·ÎºÎÅÍ NDP-sugar¸¦ °£´ÜÇÏ°Ô ÇÕ¼ºÇÒ ¼ö ÀÖ´Ù. ÀÌ ¹æ¹ýÀÇ ¶Ç ´Ù¸¥ ¹®Á¦Á¡Àº ÀϹÝÀûÀ¸·Î GT ¹ÝÀÀÀÇ °á°ú·Î »ý¼ºµÈ glycosides´Â º¹ÀâÇÑ ±¸Á¶¸¦ °¡Áö°í, ¶Ç ´Ù¾çÇÑ ±¸Á¶ÀÇ sugar¿¡ ÀÛ¿ëÇÏ´Â GT¸¦ ã±â°¡ ¾î·Æ´Ù´Â °ÍÀÌ´Ù. ±×·¯³ª ÃÖ±Ù¿¡ 2-chloro-4-nitrophenol glycosides¿Í oleandomycin D GT º¯Çüü¸¦ ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ ±¸Á¶ÀÇ UDP-/dTDP sugar¸¦ ÇÕ¼ºÇÑ ¿¬±¸°¡ º¸°íµÇ¾ú´Ù (±×¸² 4). ÀÌ ¹æ¹ýÀ» Á» ´õ È®ÀåÇÏ¿© ´Ù¸¥ NDP-sugarµµ ÇÕ¼ºÀÌ °¡´ÉÇÏ°Ô µÇ¸é glycorandomization¿¡ Å« ¼ºÀåÀÌ ÀÖÀ» °ÍÀ¸·Î ±â´ëµÈ´Ù.


4. ´ëÇ¥ÀûÀÎ glycorandomized libraries
Áö±Ý±îÁö GT¿¡ ÀÇÇÑ glycorandomization ¹æ¹ýÀ» ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ glycorandomized libraries°¡ ÇÕ¼ºµÇ¾ú´Ù. ¿©±â¼­´Â »ý¸®È°¼ºÀÌ ÀÖ´Â 2Â÷ ´ë»ç¹°À̳ª ¾à¹° À¯µµÃ¼¸¦ Áß½ÉÀ¸·Î ÀÌ·¯ÇÑ libraries¸¦ ¿ä¾àÇÏ¿© ¼Ò°³ÇÏ°íÀÚ ÇÑ´Ù.


4-1. ¿øÇü glycosyltransferases¿¡ ÀÇÇØ ÇÕ¼ºµÈ libraries
Ç¥ 1¿¡¼­´Â ÀÚ¿¬¿¡ Á¸ÀçÇÏ´Â GT¸¦ ±×´ë·Î ÀÌ¿ëÇÏ¿© glycorandomized library¸¦ ¸¸µç ¿¹µéÀ» ¿ä¾àÇÏ¿´´Ù. ÀÌ Ç¥´Â Âü°í¹®Çå¿¡ ¼­¼úµÈ ³»¿ë Áß, Áß¿äÇÑ ³»¿ë¸¸À» ¿ä¾àÇÏ¿© Á¤¸®ÇÑ °ÍÀ¸·Î, °¢°¢ÀÇ ¿¹¿¡ ´ëÇÑ ÀÚ¼¼ÇÑ ³»¿ëÀº Âü°í¹®Ç带 ÂüÁ¶Çϱ⠹ٶõ´Ù.


4-2. º¯ÇüµÈ GT¿¡ ÀÇÇØ ÇÕ¼ºµÈ libraries
Ç¥ 1¿¡¼­ ¿ä¾àµÈ ÀÚ¿¬¿¡ Á¸ÀçÇÏ´Â GT¸¦ ÀÌ¿ëÇÏ¿© ÇÕ¼ºµÈ glycorandomized library´Â ¾ÆÁÖ ¸¹Àº È­ÇÕ¹°µéÀ» Æ÷ÇÔÇÏ°í ÀÖ´Ù. ÇÏÁö¸¸, ±× È­ÇÕ¹°µéÀº ¿øÇü GT¿¡ Á¸ÀçÇÏ´Â ÇÑÁ¤µÈ ±âÁú ¼±Åüº¿¡ ÀÇÁ¸Çϱ⠶§¹®¿¡ È­ÇÕ¹°ÀÇ ±¸Á¶Àû ´Ù¾ç¼º¿¡ À־ ÇÑ°è°¡ ÀÖ´Ù. ±×·¡¼­ ¿øÇü GT¸¦ º¯ÇüÇÏ¿© º¸´Ù ´Ù¾çÇÑ ±¸Á¶ÀÇ glycorandomized library¸¦ ±¸ÃàÇÏ·Á´Â ³ë·ÂÀÌ ÁøÇàµÇ¾î ¿Ô´Ù. Áú·®ºÐ¼®¹ý20À̳ª pH¿¡ µû¶ó »öÀÌ º¯ÇÏ´Â Áö½Ã¾àÀ» ÀÌ¿ëÇÏ¿© º¯Çü GT library·ÎºÎÅÍ ¿øÇÏ´Â ±âÁú¿¡ ÀÛ¿ëÇÏ´Â GT¸¦ ¾ò¾î³½ ¿¹µéÀÌ ÀÖ´Ù. ÇÏÁö¸¸ ÀÌ ¹æ¹ýµéÀº Á¦ÇÑµÈ ½Ã½ºÅÛ¿¡ ´ëÇؼ­´Â ÀÀ¿ëµÉ ¼ö ÀÖÁö¸¸, ºñ¿ëÀûÀÎ ¹®Á¦³ª ½ÅÈ£ÀÇ ¹Î°¨µµ µîÀÇ ¹®Á¦·Î ´Ù¾çÇÑ ±¸Á¶ÀÇ library¸¦ ¸¸µå´Â µ¥´Â ÇÑ°è°¡ ÀÖ´Ù.


ÃÖ±Ù¿¡ Çü±¤¿¡ ±âÃÊÇÑ GT assay ¹æ¹ýÀÌ °³¹ßµÇ¾ú°í, À̸¦ ÀÌ¿ëÇÏ¿© ºñ±³Àû ´Ù¾çÇÑ ±¸Á¶ÀÇ Ãµ¿¬¹° À¯µµÃ¼¿Í sugar-donor·ÎºÎÅÍ glycorandomized library¸¦ ±¸ÃàÇÑ ¿¹µéÀÌ º¸°íµÇ¾ú´Ù. ÀÌ ¿¹µé¿¡¼­´Â Oleandomycin D (OleD) º¯Çü library¸¦ ¸¸µé°í, À̵é Áß ´Ù¾çÇÑ ±âÁúµé¿¡ ÀÛ¿ëÇÒ ¼ö ÀÖ´Â º¯Çü OleD¸¦ screeningÀ» ÅëÇؼ­ ã¾Æ³¿À¸·Î½á, novobiocin À¯µµÃ¼ µî ¸Å¿ì ´Ù¾çÇÑ Á¾·ùÀÇ ±Û¸®ÄÚ½ÇÈ­µÈ È­ÇÕ¹°µéÀ» ¾òÀ» ¼ö ÀÖ¾ú´Ù. ƯÈ÷ 3-3¿¡¼­ ¾ð±ÞÇÑ 2-chloro-4-nitrophenol glycosides¿Í Ole D º¯Çüü·ÎºÎÅÍ ¸¸µé¾îÁø ´Ù¾çÇÑ sugar-donor¸¦ ÀÌ¿ëÇÏ¿© GT library¸¦ screeningÇÏ¸é ±âÁ¸¿¡ ÀÖ´ø sugar-donorÀÇ ÇÕ¼º°ú assay¿¡ À־ÀÇ ÇѰ踦 ¸ðµÎ ±Øº¹ÇÒ ¼ö ÀÖÀ½ÀÌ º¸°íµÇ¾ú´Ù (±×¸² 4). ½ÇÁ¦·Î ÀÌ ½Ã½ºÅÛÀ» ÀÌ¿ëÇÏ¿© vancomycin À¯µµÃ¼ µî 11°³ÀÇ ±Û¸®ÄÚ½ÇÈ­µÈ È­ÇÕ¹°À» ÇÕ¼ºÇÏ¿´´Ù.


5. °á·Ð
½Å¾à °³¹ß¿¡ À־ ´çÀ» Æ÷ÇÔÇÑ È­ÇÕ¹°µéÀÇ À¯¿ë¼ºÀ» »ý°¢ÇÒ ¶§, À§¿¡¼­ »ìÆ캻 glycorandomized libraryÀÇ ±¸ÃàÀº ½Å¾à °³¹ß¿¡ Å« ±â¿©¸¦ ÇÒ °ÍÀ¸·Î ±â´ëµÈ´Ù. ÀÌ·¯ÇÑ library ±¸ÃàÀ» À§ÇÑ ¹æ¹ýÀ¸·Î½á, È¿¼Ò¸¦ ÀÌ¿ëÇÑ Á¢±Ù¹ýÀº ¾ÆÁÖ È¿°úÀûÀÎ ¹æ¹ýÀÌ°í, ¾ÕÀ¸·Îµµ ¸¹Àº ¿¬±¸¸¦ ÅëÇÑ Å« ¹ßÀüÀÌ ÀÖÀ» °ÍÀ¸·Î ±â´ëµÈ´Ù.

 

6. Âü°í¹®Çå
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