Rheumatoid arthritis (RA) is an autoimmune disorder characterized by systemic, erosive synovitis, and extra- articular involvement: It may result in nearly complete functional defects, lead to chronic pain and also cause chronic inflammation of the joints. The cause of RA is not yet known, but the symptoms include morning stiffness, nonspecific joint pains, swelling and tenderness around inflamed joints, and loss of functioning and mobility of joints. Copper bracelets are reported to reduce the inflammation associated with rheumatoid arthritis. It is believed that sweat transports copper through the skin to its target site. Several copper complexes possess anti-inflammatory (AI) activity and because these copper complexes almost always have stronger activity than their parent compounds or ligands, it has been hypothesized that the active form of a number of popular anti- inflammatory drugs are their copper complexes. Tripeptide Copper Complexes as Potential Anti - inflammatory Drugs for Rheumatoid Arthritis AHMED. N. H. HAMMOUDA & GRAHAM E. JACKSON Department of Chemistry, University of Cape Town, Rondebosch 7700, Cape Town, South Africa Introduction The Aim of Research Design new drugs that will alleviate the inflammation associated with rheumatoid arthritis. These drugs need to be administered dermally and be selective for Cu(II) so, that they do not affect the speciation of other metal ions in blood plasma. Copper is transported in vivo as the human serum albumin complex. Recently it has been shown that N-methylated amino acids are much more lipophilic than their un-methylated analogues. The proposed ligands combine these two ideas. NH H 2 N O HO NH N N O NH O Sarcosyl-L- histidyl-L-lysine (SKH) Results and Discussion 1. Potentiometric Titration Table 1: Stability constants (log β pqr ) for Tripeptides (SHK, SKH and SHH) β pqr = [MpLqHr]/[M]p[L]q[H]r, I = 0.15 mol.dm -3 (NaCl), T = 25 ºC. S.dev denotes standard deviation in logβ pqr ; R f H is the Hamilton R-factor and R lim H its limit Tripeptides p q r log β pqr S.dev R f H R lim H n T (n p ) SHK-H SHK-H 2 SHK-H 3 SHK-H 4 SKH-H SKH-H 2 SKH-H 3 SKS-H 4 SHH-H SHH-H 2 SHH-H 3 SHH-H 4 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 1 2 3 4 1 2 3 4 10.33 18.70 25.27 28.1 10.25 18.55 25.24 27.81 08.57 15.74 21.64 23.94 0.01 0.01 0.14 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.001 0.002 0.004 0.001 0.001 0.001 3(223) 2(130) 2(94) Fig 1: distribution curve for the Cu(II)(a)Sarcosyl-L-histidyl-L-lysine(SHK), (b) Sarcosyl-L-lysyl-L-histidine(SKH) and (c) Sarcosyl-L-histidyl-L-histidine (SHH) Complexes. Metal ion Species p q r log β pqr (S.dev) R f H(R lim H) n T (n p ) Cu(II)SHK CuLH CuL CuLH -1 CuLH -2 1 1 1 1 1 1 1 1 1 0 -1 -2 20.06(0.03) 15.54(0.03) 06.10(0.04) -4.55(0.04) 0.02(0.003) 4(202) Table 2: Stability constants (log β pqr ) for Cu(II) with Tripeptides (SHK, SKH and SHH) β pqr = [MpLqHr]/[M]p[L]q[H]r, I = 0.15 mol.dm -3 (NaCl), T = 25 ºC. S. dev denotes standard deviation in logβ pqr ; R f H is the Hamilton R-factor and R lim H its limit Cu(II)SKH Cu(II)SHH CuLH CuL CuLH -1 CuLH -2 CuLH CuL CuLH -1 CuLH -2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 -1 -2 0 -1 -2 1 17.20(0.30) 12.96(0.04) 07.55(0.03) -2.80(0.05) 16.40(0.02) 12.01(0.02) 05.27(0.03) -3.78(0.04) 0.01(0.002) 0.01(0.003) 2(178) 2(152) Species SHK Experimental λ max (nm) ε max (M.cm-1) Calculated λ max (nm) Possible donor atoms CuLH CuL CuLH -1 CuLH -2 SKH 610 607 577 575 67.94 61.39 85.05 67.47 602 602 576 576 -NH, N- , Nim,H 2 O -NH, N- , Nim,H 2 O -NH, N- , Nim,OH- -NH, N- , Nim,OH- 2. Ultraviolet-Visible Spectroscopy Table 3: Visible spectrophotometric Ɛ max (M.cm-1)and λ max (nm) Experimental and Calculated values, together with possible donor groups for Cu(II) with Tripeptides (SHK, SKH and SHH) Complexes CuL CuLH -1 CuLH -2 SHH CuLH CuL CuLH -1 CuLH -2 525 525 525 630 600 545 525 80.84 146.84 151.78 47.74 82.74 103.72 142.63 531 531 531 622 602 576 536 -NH, N- , N-, Nim -NH, N- , N-, Nim -NH, N- , N-, Nim -NH, N- , Nim,H 2 O -NH, N- , Nim,H 2 O -NH, N- , Nim,H 2 O -NH, N- , N-, Nim, Fig 2: Molar extinction coefficients a function of λ max for Cu(II)-(a) Sarcosyl-L-histidyl-L-lysine (SHK), (b) Sarcosyl-L-lysyl-L-histidine (SKH) and (c) Sarcosyl-L-histidyl-L-histidine (SHH) Complexes. 3. 1 H NMR spectroscopy NH H 2 N O HO NH N N H O NH O Sarcosyl-l-histidyl-l-lysine (SHK) a b c d e f Fig 3: Raw 1H NMR titration for the complexation of Sarcosyl-L-histidyl- L-lysine (0.075M) with Cu(II) (0.0118M) in D 2 O:H 2 O 1:9 mixture. Fig 4: Raw 1 H NMR titration for the complexation of Sarcosyl-L-histidyl- L-histidine (0.11 M) with Cu(II) (0.0118M) in D 2 O:H 2 O 1:9 mixture. 4. Blood-plasma simulation studies Fig 5: Cu(II) plasma mobilizing index for SHK1, SKH2 and SHH3. CuL (SHK) CuLH -1 (SKH) CuLH -1 (SHH) The blood plasma model, predicted that the Cu(II)-SHK, Cu(II)-SKH and Cu(SHH) complexes are able to mobilize Cu(II). Fig 6: Possible structures of the CuL species (SHK) and MLH -1 species (SKH & SHH) at pH 7 calculated by studio discovery. Conclusion The Cu(II) tripeptide complexes (SHK, SKH and SHH) showed a significantly different coordination behavior in acidic/neutral and basic pH-range. The type of coordinate of SHK and SHH by N-terminal amino group, deprotonated amide, the N 3 -imidazole nitrogens and the water molecular. SKH was found that coordinate equatorially through the imidazole ring of His, the N-terminal amino group and the two amide nitrogens existing between them. 0 20 40 60 80 100 2 4 6 8 10 % Cu(II) pH Cu(II) MLH ML MLH-1 MLH-2 C NH N H N O HO NH N N H O NH O Sarcosyl-L-histidyl-L-histidine (SHH) a2 b2 c d a1 b1 c d e f 0 40 80 120 160 400 500 600 700 800 Ɛ max (dm 3 .mol -1 .cm -1 ) Wavelength (nm) Cu(II) CuLH CuL CuLH-1 CuLH-2 C