TAP CHi HOA HOC T. 50(2) 193-195 THANG 4 NAM 2012
T 6 N G Hgp THu6c THLT DUNG TRONG PHAN TICH
CAC Hgp CHAT THIOL
Tran Thj Thu Thuy'*, Ngd Nggc Thang', Tran Quoc Toan', Nguyen Quyet Chien', Phan Dai Lam^
Vi^n Hoa hgc, Vi^n Khoa hgc vd Cdng ngh4 Vi^t Nam 'Cgc Ky thugt, Binh chung Hda hgc
DenTdaso^in 14-10-2011
Abstract
EUman's test is an effective method for the determination of free thiols, and is applied in many fields. However, a few reagents are used for this test. In this report, uc discuss a reliable route to synthesize an Ellman's reagent reported by Veskler which is very usefull but commercially not available.
Keywords: Ellman's reagent, thiols, disulfite.
1. GIOI THIEU
Thuoc thu EUman (5,5'-diihiobis-(2-nitrobenzoic acid), ggi tdt Id DTNB) dugc su dung de xdc djnh cdc thiol tu do trong mdt mdu bang phucmg phdp so mau [1]. Phuong phap ndy dugc irng dyng trong
nhieu ITnh vuc nhu cdng nghiep, ndng nghiep, y te, mdi trudng ciing nhu trong nghien ciiu khoa hgc.
Nguyen ly ciia phuong phap nay dua tren phan irng cua thiol RSH vdi thudc thu DTNB, chia cdt lien ket disulfit, tao ra axit 2-nitro-5-thiobenzoic (NTB). Trons mdi trudng kiem, NTB phdn ly thdnh dianion NTB " cd mdu vdng.
i s ^ - ^ - Y
Nong dg cua chat phdn tich dugc xdc djnh dua thdp, khdng vugt qud 20800 M"'cm" cd mau nhgt.
tren ndng do eua NTB'', dugc xac dinh blng quang Do do, phep do kem v^ do nhay va khd bang mdt phd hdp thu tgi budc sdng 412 nm voi he sd hdp thu thudng.
14150 M"'cm"'. D^ kh4c phyc cac nhuge di6m tren, Veskler vd Ngodi DTNB, mgt sd cac chat khac nhu 2,2'- vd cgng su da nghien ciru vd tdng hgp ra hgp chdt mau 4,4-dipyridyl disulfit cung dugc sir dung [2]. Tuy azo cd cdu true nhu sau [3]:
nhien, cdc chat mdu tren cd hgn che la he sd hap thu .Cl
OH NaOgS
"^-(Z \ ) _ N = N - ( \ / > - S - S - < v / > - N = N H f ' h ^ (1) NaOsS
Chat mdu ndy tan tdt trong nudc vd cdc dung mfii hu'u CO phan cgc. Dung djch ciia hgp chdt ndy cd eye dgi hdp thu d 515-535 nm, vdi he sd hdp thu cao (70000 M''cm''), sdn phdn tao ra co cue dgi hap thu d 580-595 nm (35500 M"'cm"'), 600 nm (34500 M"'cm"'). Do dd, dO nhgy cua phuong phdp tdng, dl ddng thuc hi?n dugc bdng mdt thudng.
Thudc thir mdi nay dd dugc irng dyng trong
phuong phdp nhdn biet nhanh cdc chat dgc thdn kinh dua tren su Ore che men axetylcholinesterase (AChJE) [4], Id e n ^ m xuc tac cho s\r thiiy phan chdt dan truygn than kinh axetylcholine tgo ra thiocholine.
Chat mau azo (1) khong cd ban tren thj trudng va chi cd mgt tai lieu tham khao ciia Veksler lien quan tdi hgp chat nay. Do dd, chung to! de ra muc tieu nghien ciru vd xdy dung qui trinh tdng hgp hgp
TCHH, T. 50(2), 2012
chat nay tir cac nguyen MQU dau cd bdn tren thj trudng.
2. THUC NGHIEM
Phuong phdp chung: Pho hong ngogi FTIR dugc ghi tren mdy Impact 410. Pho khoi ESI-MS dugc ghi tren may LC/MSD Agilent Series 1100. NMR dugc ghi lien nia> Bruker Avance 500 Mil?,
Hoa chat: I Ida chat dung cho phan irng mua ciia hang Aldrich vd Merck. Dimg moi diing cho cdc myc dich khac Id logi diing cho long hgp hda hgc ciia Trung Quoc. dugc cat l^i trudc khi dimg. Chat chuan dugc cung cap tir Cyc ky thugt-Binh chung Hda hgc. Sdc ky cgt su dyng chat hap phy Id Silica gel 60 (0.015-0,040 mm) for column chromatography cua Merck. Sdc ky Idp mdng sir dyng bdn dc nhdm trdng sdn silica gel Merck 60 Visi- Thuoc hien mau la CeSOj.
Axit 4-choro-5-hydroxynaphlhalen-2,7- disulfonic 4' Hdn hgp gdm 5,0 g axit mononatri 4- amino-5-hydroxynaphthalen-2,7-disuironic (2) (14,7 mmol), 0,587 g NaOH (14,7 mmol) vd 1,035 g NaNO: (15 mmol) dugc hda tan trong 10 ml nudc.
Lam lanh hdn hgp thu dugc xudng 0°C vd khuay deu. Sau dd, nhd gigt 6 ml HCI bdo hda vao hdn hgp, duy tri nhiet do ciia hdn hgp phdn irng d 0-5°C.
Hdn hgp phdn irng ddn ddn dac tai va chuyen sang mau vang dam. Sau khi nhd gigt xong HCI, khudy hdn hgp phdn iirng trong 1 gid. Giir nhiet dg ciia hdn hgp sau phdn irng (mudi diazo 3) dudi S^C.
Chudn bj dung dich cua 1,5 g CuCI (15,3 mmol) trong 5 ml HCI bdo hda vd lam Ignh xudng O^C.
Khudy deu dung dich vd cho tir tir muoi diazo long hgp dugc d tren vdo. Sau dd, tang nhi?t dg cua hdn hgp phan irng len 10°C. Hdn hgp phan iing dan ddn chuyin sang mau tim va cd bgt khi Ni thodt ra.
Khudy hdn hgp phdn irng cho den khi het bgt khi.
Cuoi eiing, tang nhi?t dg cua hdn hgp len 70''C vd them 10 g NaCI de lam ket tiia sdn pham. Lgc lay sdn pham vd rua bang dung djch NaCI bao hda. San phdm sau khi da Idm khd cd ldn mgt it NaCI, khong dnh hudng tdi khd nang phdn irng sau nay khi tham gia phdn iing ghep ddi. Hi?u suat dgt khodng 55%.
IR (cm'): VOH: 3505,30 (m); x>„p: 1645 (lb);
1575 (tb);u„c-s: 1367,91 (tb);o3C-s: 1189,45 (m), UQ:
1049,02 (m).
'HNMR£ii,p„(CD30D): 8,2305 (IH, d, 7 = 1,5 Hz; H-3); 7,917 (IH, d,J= 1,5 Hz; H-1); 7,885 (IH, d,J= 1,5 Hz; H-8); 7,395 (IH, d,J= 1,5 Hz; H-6).
'^C NMR (5pp^ (CD3OD): 155,9 (C-5); 145,5 (C-7); 144,2 (C-2); 137,2 (C-9); 131,7 (C-4); 127,3 (C-l); 126,5 (C-3); 123,9 (C-8); 119,0 (C-10); 110,6 (C-6).
Trdn Thj Thu Thiiy vd cgng sir OH Cl
HO3S MS (m/z): [M+] 337,5
SO3H
Natri 3.3'-(4.4'-di.sulfanediylbis(4,l-phenyl ene)bis(diazen-2,l-diyl))bis(5-cloro-4-
hydroxynaphthalen-2,7-disulfonal) 1: Hda tan 0,2 g 4/r-diaininodiphcnyl disulfii (5) (0,81 mmol) trong 1.2 ml HCI bdo hda vd 5 ml nudc. Sau do, lam lanh hon hgp xuong 0"C vd khuay deu. Them vdo Ii6n hgp dung djch gom 0,112 g NaN02 (1,62 mmol) trong 3 ml nudc de diazo hda 4,4'-diaminodiphenyl disulfii, Sau khi them het dung djch NaN02, hon hgp phdn irng chuyen thanh mdu vang nhgt, khuay licp 30 phiit d 0-5"C de phan irng ket thuc.
Chuan bj dung djch gom axit 0,55 g 4-cloro-5- hydroxynaphlhalcn-2,7-disuIfonic (3) (1,62 mmol) vd 0,1 g NaOH trong 5 ml nudc vd ldm Ignh xudng 0"C. Khuay deu dung djch vd Ihcm tir tir dung djch muoi diazonium tdng hgp dugc d Ircn vdo. Nhi|t Ai) phan irng dugc duy tri d khodng 5-7''C. Mdu ciia hon hgp phdn irng chuyen dan sang dd dam. Khuay tiep hdn hgp phdn ling them 30 phiit nira rdi them 6 ml dietyl etc vdo, lgc Idy ket tiia va tinh che bdng sdc ky cgt (etyl axetat:/)-hexan = 2 1). Hi?u sudt phdn iing dgt 41%.
IR (cm"'): UoH 3446,01 (m); u„p 1630,33 (tb);
1491,94 (m); UN=N 1571,78 (y); o ^ . s 1382,83 (y);
u,c-s 1196,55 (m);\)ci 1054,18(m).
'H NMR (!\^^(CDjOD): 8,07 (2H, d, J = 1,5 Hz;
H-8, 8'); 7,91 (2H, d, ^ = 1,5 Hz; H-6, 6'); 7,84 (4H, d , y = 9 H z , H-12, 12', H-16, 16'); 7,77 (2H,s;H-1, 1'); 7,65 (4H, d , y = 9 Hz; H-13, 13', H-15,15').
'^C NMR <itp„, (CDjOD): 150,4 (C-7, 7'); 143,7 (C-14, 14'); 143,2 (C-2, 2'); 139,7 (C-9, 9'); 136,9 (C-4. 4'); 136,1 (C-11, 11'); 130,9 (C-13, 13', IS, 15'); 129,9 (C-10, 10*); 129,5 (C-6, 6'); 129,0 (C-3;
3'); 126,9(C-8, 8 ' , 5 , 5'); 123,1 (C-l, 1'); 119,9(C- 12, 12', 16, 16').
JpSOsNa
MS: pic phdn tir 1034,3.
3. KET QUA v A T H A O LUAN
Chien luge long hgp cua chung tdi dua tren phan ung ghep azo. Qui trinh phdn irng dugc md ta tren sff do sau, trong dd cau tii diazo la chat (6) vd ciu tii azo la chat (4):
Tdng hgp thuoc thu dimg trong...
OH Cl (4) SO3H
Cau tir azo (4) d u g c tdng h g p tir a \ i l mononatri 4-amino-5-hydroxynaphtlialen-2.7-disuIfonic (2) theo phdn iing Sandmeyer. Phdn irng xdy ra qua hai giai dogn, trong giai dogn ddu d u n g djch phan u'ng ddn ddc lai, do dd hon h g p phan irng can phdi d u g c khuay tdt. Ket thiic phdn iing thu d u o c mudi diazo (3). mudi nay phai d u g c giu" d nhiet d o d u d i 5°C de tranh bj phan huy. Chiing tdi iha\ nen do hdn h g p cua mudi diazo (3) vdo d u n g djch CuCI trong HCI nham tranh khd nang phan iing ciia mudi diazo vdi san pham the elo (4) vira d u g c tao ra. Khi phdn irng Sandmeyer ket thuc, NaCI d u g c them vdo d u n g djch nong de gidm d o tan ciia san phdm. lam ket tiia san phdm. Dung dich sau dd d u g c Idm lanh rdi lgc, tuy nhien, hat ket tiia nhd vd mjn, gay khd khan khi Igc va mdt rdt n h i l u thdi gian. Chiing toi da t h u p h u o n g phap lgc ndng, qud trinh lgc rdt de d a n g vd thudn tien. Do dd, tuy Igc lanh hieu sudt thu d u g c cao hon, nhung vi khd khan va bdt lien khi lgc nen chiing tdi chgn phuong phdp lgc ndng.
Cdu tii diazo (6) d u g c tdng h g p tir 4 - aminophenyl disulfide d u g c bang phan irng diazo hda. T i l p do thuc hien phdn iing g h e p ddi, d o mgt phdn tii mudi diazo (6) phdn irng vdi 2 phdn tii natri 4-cloro-5-hydroxynaphthalen-2,7-disulfonat (4), do vdy nen dd tir tir mudi diazo (6) vdo d u n g djch ciia natn 4-cIoro-5-hydroxynaphthaIen-2,7-disulfonat
(4). Nhiet dg phan ii-ng ghep ddi phdi duy tri d khoang 5-7^0, neu nhi?t dg tang len 10''C, co sy xuat hien khi N2 do su phdn hiiy ciia muoi diazo.
Cac ddc trung hda ly cua sdn phdm d u g c trinh bay trong phdn t h u c nghiem. Ket qua do phd tii ngogi kha kien ciia hgp chat ndy cd 4 pic tai cac b u d c sdng 532, 512, 305 vd 246 nm.
4. K E T L U A N
Chimg tdi da long h g p thdnh cdng chat mau 1.
Cau triic ciia san phdm d u g c khdng djnh bang cac p h u o n g phap vdt ly hien dai. Quy trinh tdng h g p cd tinh lap lai cao vd hieu suat dn d m h , cd the t h u e hien vdi quy m o Idn.
T A I LIEU T H A M K H A O
1. G. Ellman. Archives of Biochemistry and Biophysics, 82,70-77(1959).
2. K, Veksler, E. Golubera, A. LaVrent'ev. USSR SU 1277627(1986).
3. K. V. Veksler, C. M Trakhnova USSR SU 1177296 (1984).
4. Fabiana Arduini, Aziz Amine, Danila Moscone, Francesco Ricci & Giuseppe Palleschi. Anal.
Bioanal. Chem., 388, 1049-1057 (2007).
Lien h4: Tran Thi T h u T h u y Vien Hda hgc
Vien K h o a hgc vd C o n g nghe Vi?t N a m 18 Hodng Q u o c Viet, Cdu Gidy, Hd Ngi Email: [email protected].
