AR EFFICIENT HETHOD FOR C~NJ~~~I~ OF THIAMINE TO PROTEINS

iI. Jayamani and Philip S. Low Chemistry Department, Furdue University, W. Lafayette, fN 47907

(Received 10 April 1992) ABSTRACT: New derivatives of thiamine were prepared by reacting auccinie, glutaric or ~aleic anhydride with the hydroxyl group of thiamine. The resulting free earboxyl group was then activated using N, N, N-,N'-tetr~e~yl-O-(-N-succini~~dyl)uron~~ tetrafluoroboratel 3 and conjugated to proteins. Alternatively, proteins were reacted with P-iminothiolane and the resulting sulfhydryl groups were then added across the double bond of thiamine monomaleiate 2c prepared by the above method.

Recently, we reported fhatvitruefnreccsptor-mediated endocytosis eanbe exploited to deliver

macromolecules nondestructively into living calls if the macromolecules are first covalently

linked to folic acid2*(a members of the; vitamin B family) or biotin=. This observation

prompted us to explore thiamines. an essential coenzyme and a member of the vitamin B family,

as a possible ligand Eo mediate the uptaka of macromolecules into cells and tissues

expressing cell surface thiamine receptors. The present paper describes new simple methods

for conjugation of thiamine to proteins.

1 2

MA

1007

Cur strateS3'(Scb@se-l) was to open a cyclic s~ydr~de 1 using th8 hydroxyl group of

thi~i~e as a nuc~eoph~~e. The ~rivedprod~~t contains not only a free caxboxyX at the open

end which can be finked to a protein, but also the ester linkage ta thiamine which could

conceivably be readily hydrolyzed by esterases after entry of the protein into the cell.. In

addition, the long alfphatic spacer contributed by the a~~d~~de should pro&de better

separation of thiamine frosn the protefn and allow improved racognftlon of the vitamin by its

cell surface receptor.

Attempts to fena t&famine monosucc~~te 2a by direct he&ting(PO*C} of thiamfne

c~loride.~Cl with auccinfc anhydride, ox by heati~g(9O.C~ the same reaction components in

pyrfdfne did not yield any isolable product. However, when a suspension of tbiamfne

chlorfde.HCI (3.37 g% 10sneole) in pyridine (50ml) was heated (80°C) with succinic anbydxfde

(5 g, 50 mmole) and 4-(~,~-dimethyl)~in~p~idine (1.22 g, 20 mmole), the product thiauine

monosuccinatek 2a was obtained in excellent yield (3.2 g, 0001, Tbe product was filtered,

washed with acetone, and recrystallized fron 95% ethanol (mp,180-5*C decam.)

Lsnkage to a protein was as foflows: To a suspension of thiamine monosuccinate 2s

(1.28 tng, 2.94 .mola) in DMSO (100 PL), N,N,N~,N'-tetrslset-hyl-O-(-N-auccinimfdyl)uroni~

tetrafluoroborate 4 (0.88 mg, 2.94 pxole) and triethylamine (0.59 mg, 5.88 jmole) were added

and stirred fox 15 min. The above laixturs was than added slowly to a solution of bovine

serum albumin (WA) (10 mg, 0.147 mole) in phosphate buffered saline (PBS) (I mL) pN 7.

After lb, the thi~i~e labeled BSAwas separated from other components and the @was changed

to 5 by passing the mixture tbrougb a Iczn x XOcm Sephadex-MS desalting column. BSA

concentration was than estimated by the BCA *ethod5. ~i~ina attached to BSA wae

estinated6 (3 thi~~nes per BSA) by oxidizfng the conjugated tbia&ne to thiccbrome and

naeasuring the level of fluorescence at excitation 365 ND and erafssion 445 ruu. For this

purpose, s standard curve was constructed frost the fluorcrscence of a known amount of thiamine

aixed with the same, mount of unmodified BSA sample. The low extent of BSA derivatizatfon

my have derived iron! tha instabbflity of the activated thiamine aster in aqueous solution or

from the tendency of hydropb~bie molecules to bind noncovale~tly to lipophilic sites on BSA.

The poor solubility of thiamine monosuccinate 2a in DMSO prompted us to look for a

still better derivative. Thus, thfamine ~o~oglutarate 2b was prepared in 85%(3-S g) yield

Efflcicntconjugationofthi~inetoprotcins 1009

by stirring and hasting (80.C) a suspension of tbismfne chlorlde.HGl (3.37 g, 10 mmole) in

pyridine (50 s&aL) with glutattc mnbydride (5.7 g, 50 amrole). Tha product7 2b was purified

(~qp, 200°C decom,) us described for 2a. It msy be worth nantioning that the presence of

4-(N,N-dimgthyl)lippincpyrfd~ne led to decomposition of the product and glutaric anhydride.

Thiamine ~noglu~arate 2b was activetad and reacted with BSA as in 2a and the resulting

thlemine content (4 thiamine per BSA) was estimateds,

WA-NH2 +

4

Schsme - 2

A large number of proteins have su~~yd~~ groups and those lacking thiols can be

easily modified to acquire them(Scheme-2). Such sulfhydryl groups can then be easily added

to a reactive double bonda. To exploit this conjugation chemistry, thismine monomaleiatee2c

was prepared in 83%(3.3 g) yield by heating (SS'G) and stirrfng(3 hr) a mixture of thiamine

chloride.HCl (3.37 g, 10 mmole) and maleie anhydride (4.9 g. 50 asmole) and the product was

purified (mp 230.2'C decom.) as 23, To test the proposed reaction (Scheme-3). thiamine

monomaleiate 2c (0.4 g, Z mmole) was reacted with Z-aminoethanethiol.HC~ CC.114 g, 1 mole)

in 25% aqueous methanol (5 ml) at 25'C for 12 h. titer methanol removal by rotary-

evaporation and crystalliaation in VS% ethanol, the product 2 or 3-(2r-~~noe~ylthio)

4-(thismine)monosuccinate 5 lo (mp 165.7O'C decom.) was obtained quantitutively. However,

no attempts were made to find the ratio of the positional isomers.

1010

NH2 cl-

P/

A' 3 =2\+

N% Cl'

WJ-C?

v

HStXi,M,Bli,.HCl NH

\ .’ “r

rnz\+

wA

N_

\ SQJ2GB2BH2.HCl

9 N

a3 as N

k

XS

cH,-cH,-o-co-~--~ =H, I

cH,-cHH,-o-co-cH,-CIf-COOH

2c

Scheme - 3

+

BSA (10 mg, 0.145 /mole) in PBS (pH 8) was reacted with iminothiolane.HCl 4

(2.94 wale) for 3 h at 25'C. Unreacted iminothiolane was removed and the pH was adjusted

to 5 by passing the above reaction mixture through a 1 cm x 10 cm Sephadex-G25 desalting

column. The derivatized BSA was treated immediately with thiamine monomaleiate 2c (1.17 mg,

2.94 mole), and after 3 h the BSA was purified and the number of thiamines per BSA was

estimated'(3:l).

In conclusion, we have reported a simple method to attach thiamine to any protein, thus

opening a path to a number of biological investigations. Details regarding thiamine-mediated

delivery of macromolecules into living cells will be reported elsewhere.

Acknowledgment: We gratefully acknowledge a research grant from Eli Lilly.

Efficient conjugation of thiamine to proteins 1011

References and Notes:

1.a. Bennwarth, W.; Schmidt, D.; Stellard, L. It.; Hornung. C.: Knorr, R. and Miiller, F.;

Xelv. Chew Acta. 1988. 71, 2085.

b. Knorr, R.; Trzeeiak, A.; Bannwarth, W. end Gillessen. D.; Tetrahedron lett. 1989, 30,

1927.

2.e. Leamon. C. P. and Low, P. S.; Proc. Natl. Acad. Sci. USA. 1991, 88, 5572.

b

3.

4.

5.

6.

7.

Horn, N. A.; Neinstein, P. F. end Low. P. S.; Plant Phys. 1990, 93, 1492.

Kluger, R.; Chem. Rev. 1987. 87. 863,

For 2e. 1H-NMK(200HHz,DzO) 6 (overlapping two s,6H 6 a e1,4H)2.4-3.2, (t,2H)3.5-3.7,

(t.2H)4.5-4.7, (s,2H)5.5, (s,lH)7.9. (s.lH)9.7. 1sC-~(200~z, D20) 6 13.67, 24.38,

28.43, 28.43, 33.61. 53.09. 66.29, 108.33, 138.29, 146.49, 151.138, 165.8, 165.81.

167.85, 178.28, 182.71. IR(nujo1) cm“ 3280, 1745. 1660, 1595, 1560, 1210, 1155.

U V(water) Emax.l2238/237nm. C1.H' 365.1276 (Calcd. for C1&21NI0,S* 365.1284)

Smith, P. K .; Krohn, R.I.; Hermenson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzeno,

M. D.; Fujimato, E. k.; Coeke, 1. K.; Olson, B. J. and Klenk, D. C.; Anal. Biochem.,

1985, 76, 150.

The United States Pharmacopeie xxth rev, United State Pharmacopeia Convention, Inc.

Rockvilla, HD., 1980, p.928.

For 2b. 1H-NMR(200MHz,DzO) 6 (q,2H)1.7-1.9, (overlapping two s,6H & a m, 48)2.3-2.5.

(t,2H) 3.3-3.4, (t,2H)4.2-4.3. (s,2H)5.5, (s.lH)7.9, (s,lH)9.6. 13C-NMR(200MHz,DzO)

6 13.67, 22.13, 23.59, 28.45, 35.44, 35.55, 52.8, 68.22, 108.8S, 138.52. 146.53.

147.77. 157.96, 166.15, 166.36, 178.38. 180.81. IR(nujol)c& 3200, 1730, 1650. 1600,

1525, 1210. 1155; U V(water) Emax. 15104f243n1s. 12155/26Onm. FM.@ 379.1434 (celcd.

for Cx1Hz3N,0& 379.1440)

8.a. Bletter, W. A.; Kuenzi, S. 8.; Lambert, J. M. and Senter. P.D.; Biochemistry 1985. 24.

1517.

b. Partis, M. D.; Griffiths, D. G. end Roberts, G. J.; Pro. Chem., 1983, 2. 263,

1012 M.JAYAMANI and P.S.Low

9. For 2c. 'H-NMR(200KHz.D~O) 6 (s,38)2.48, (s,3H)2.56, (t,3H)3.3-3.4, (t,2H)4.4-4.5

(s.2HI5.5 (d,lH)6.1-6.2, (d.lH)6.5-6.5, (s,lH)7.9, (s,lH)9.6.'SC-NHR(200MHz,D,0) 6

13.63, 23.52, 28.36, 32.77, 66.91, 108, 129.7, 136.1, 138.33, 146.7, 147.69, 157.98,

166.11. 166.76, 169.7, 173. ZR(nujol)cm -= 3210, 1715, 1745, 1655, 1655, 1645, 1605,

1595, 1515, 115s. U V(water)Emax. 11117/242nm. 10643/26Onm. FAR.@ 363.1105 (calcd.

for C1,HI&O,S+ 363.1127)

10. For 5. 1H-NblR(200NHz,D,0) 6 (s,3H)2.48, (s,3H)2.55, (m,4H)2.7-3, (m,4H)3.1-3.3,

(m,lH)3.6-3.6, (t.2H)4.4-4.2, (s,2H)5.5, (s,lH)7.9, (s,lH)9.6.1%-NMR(200NRz,D~O) 6

13.74. 23.63, 28.47, 31.05, 41.13, 44.33, 52.82, 66.71, 108.82, 138, 146.2, 147.83,

157, 161.15, 166.36, 175.34, 178.44. XR(nujol)cm -I 3200, 1720, 1730, 1650, 1605, 1305.

1150. UV(water) Ehax. 10989/242nm, 12192/26Onm. FAB.ti440.1409 (calcd. for C,,H,@,O,S,t

440.1426)