Evidence for species-specific substrate-site-directed inactivation of rabbit adenylate kinase by N6-(6-iodoacetamido-n-hexyl)adenosine 5'-triphosphate

Abstract

Adenosine 5'-triphosphate (ATP) derivatives bearing iodoacetylamino-n-alkyl substituents [(CH2)nNHCOCH2I] on N6 were synthesized as potential ATP-site-directed irreversible inhibitors of adenylate kinases from rabbit, pig, and carp muscle. When n was 5 no enzyme was progressively inhibited (inactivated) by 1 mM inhibitor under the test conditions (6 h at 0 degrees); when n was 6 the rabbit enzyme was 76% inactivated by 0.79 mM inhibitor whereas the pig and carp enzymes were unaffected by 2.76 mM inhibitor; when n was 7, 1 mM inhibitor inactivated 14% of the rabbit enzyme and did not inactivate the pig and carp enzymes; when n was 8, all enzymes were inactivated 11-15% by 1 mM inhibitor. No inactivation occurred when the iodine of the hexamethylene analogue was replaced by hydrogen. The selective effect occured also in mixtures of the rabbit and pig enzymes and evidence could not be found that the hexamethylene analogue was activated by the rabbit enzyme or deactivated by the pig and carp preparations. The species-specific inactivation in concluded from various lines of evidence to be ATP-site-directed and is attributed to alkylation of an amino acid residue of the rabbit enzyme which in the pig and carp enzymes is absent, inaccessible, or less reactive. These and previous studies with several other enzymes provide evidence that substrate-site-directed agents capable of bonding covalently to an amino acid residue outside the substrate site can be designed to exert species-specific or tissue-specific irreversible inhibition of target enzymes.

N-Tritioacetoxyphthalimide: A New High Specific Activity Tritioacetylating Reagent

“…B. N -Acetoxyphthalimide. Previous syntheses of this compound were not well described. ,, The current synthesis is similar to early preparations of N -(iodoacetoxy)succinimide. ,, N -Hydroxyphthalimide (0.7 g, 4.35 mmol), acetic acid (HOAc, 258 μL, 4.35 mmol), and dicyclohexylcarbodiimide (DCC, 0.85 g, 4.35 mmol) were added to dry ethyl acetate (EtOAc, 125 mL), and the mixture was stirred for 3 h at room temperature and then filtered. The filtrate was dried (Na 2 SO 4 ), and the residue was crystallized from ethanol to give a white solid (0.82 g, 90%): mp 185 °C; 1 H NMR δ (CDCl 3 ) 7.76−7.89 (m, 4H), 2.39 (s, 3H) …”

“…B. N -(Iodoacetoxy)phthalimide. This synthesis was based on published preparations of N -(iodoacetoxy)succinimide. ,, N -Hydroxyphthalimide (1.4 g, 8.7 mmol), iodoacetic acid (1.6 g, 8.7 mmol), and DCC (1.7 g, 8.7 mmol) were added to dry EtOAc (250 mL), and the mixture was stirred for 5 h at room temperature. The reaction mixture was then filtered, the filtrate was dried, and the residue was crystallized from ethanol to give a white solid (2.15 g, 75%): mp 120 °C; 1 H NMR δ (acetone- d 6 ) 8.01 (m, 4H), 4.32 (s, 2H).…”

“…C. N -(Iodoacetoxy)naphthalimide. This synthesis was based on published preparations of N -(iodoacetoxy)succinimide. ,, The sodium salt of N -hydroxynaphthalimide was acidified and yielded two products, with the major component being the ring-opened product. N -Hydroxynaphthalimide (22 mg, 0.1 mmol), iodoacetic acid (18 mg, 0.1 mmol), and DCC (19.5 mg, 0.1 mmol) were added to dry EtOAc (2 mL), and the mixture was stirred for 5 h at room temperature.…”

“…The analogous N -acetoxy derivatives could be expected to be equally reactive, but have been infrequently utilized. 2,4,7,10-12 Specifically, N -acetoxyphthalimide was used to prepare N -acetylmuramic acid, , N -(iodoacetoxy)succinimide was used for iodoacetylation of N 6 -(6-amino- n -hexyl)adenosine 5‘-phosphate, and tritiated acetyl derivatives have been produced at low specific activity. , De Groot 10 reacted [ 14 C]phenylalanyl-soluble-RNA with N -acetoxysuccinimide and N -(tritioacetoxy)succinimide (no specific activity indicated), and Lindsay 2 used N -(tritioacetoxy)succinimide at 21 GBq/mmol to tritioacetylate insulin. In a single example, a high specific activity tritioacetyl group was made by iodoacetylation of muramyl dipeptide with N -(iodoacetyl)succinimide and subsequent catalytic tritiodehalogenation …”

Reactive 5'-substituted 2',5'-dideoxyuridine derivatives as potential inhibitors of nucleotide biosynthesis

“…This procedure is an improvement over a previously described conversion of 2 to 7 via the 5-mercuriacetate.12 Reaction of 7 with 4-nitrophenyl bromoacetate by the usual procedure gave an 85% yield of the bromoacetamide 8. The corresponding 5'-amino-2',5'-dideoxy-5-iodouridine (9) was prepared from 2 via the 5'-mercuriacetate12 and converted in 93% yield to the bromoacetamide 10. A similar reaction of 5'-amino-2',5'-dideoxy-5-fluorouridine13 (1 1) with 4-nitrophenyl bromoacetate gave a 63% yield of 12.…”