1.A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.
Kodama K1, Fukuzawa S, Sakamoto K, Nakayama H, Kigawa T, Yabuki T, Matsuda N, Shirouzu M, Takio K, Tachibana K, Yokoyama S. Chembiochem. 2006 Oct;7(10):1577-81.
An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.
2.Enantiomeric differentiation of β-amino alcohols under electrospray ionization mass spectrometric conditions.
Karthikraj R1, Chitumalla RK, Bhanuprakash K, Prabhakar S, Vairamani M. J Mass Spectrom. 2014 Jan;49(1):108-16. doi: 10.1002/jms.3312.
The enantiomeric differentiation of a series of chiral β-amino alcohols (A) is attempted, for the first time, by applying the kinetic method using L-proline, L-tryptophan, 4-iodo-L-phenylalanine or 3, 5-diiodo-L-tyrosine as the chiral references (Ref) and Cu(2+) or Ni(2+) ion (M) as the central metal ion. The trimeric diastereomeric adduct ions, [M+(Ref)2+A-H](+), formed under electrospray ionization conditions, are subjected for collision-induced dissociation (CID) experiments. The products ions, formed by the loss of either a reference or an analyte, detected in the CID spectra are evaluated for the enantiomeric differentiation. All the references showed enantiomeric differentiation and the R(chiral) values are better for the aromatic alcohols than for aliphatic alcohols. Notably, the R(chiral) values of the aliphatic amino alcohols enhanced when Ni(2+) is used as the central metal ion. The experimental results are well supported by computational studies carried out on the diastereomeric dimeric complexes.
3.Regioselective carbon-carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry.
Kodama K1, Fukuzawa S, Nakayama H, Kigawa T, Sakamoto K, Yabuki T, Matsuda N, Shirouzu M, Takio K, Tachibana K, Yokoyama S. Chembiochem. 2006 Jan;7(1):134-9.
Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki-Heck reaction for site-specific carbon-carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6-fused Ras protein containing 4-iodo-L-phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography-mass spectrometry (LC-MS). The regioselectivity of the Mizoroki-Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS.
4.p-ethynylphenylalanine: a potent inhibitor of tryptophan hydroxylase.
Stokes AH1, Xu Y, Daunais JA, Tamir H, Gershon MD, Butkerait P, Kayser B, Altman J, Beck W, Vrana KE. J Neurochem. 2000 May;74(5):2067-73.
Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in serotonin biosynthesis. The enzyme activity is dependent on molecular oxygen, a tetrahydropterin cosubstrate, and ferrous iron. The present study demonstrates that TPH is inhibited by a novel compound, p-ethynylphenylalanine (pEPA), produced by the Heck reaction of trimethylsilylacetylene with N-tertbutyloxycarbonyl-4-iodo-L-phenylalanine methyl ester. pEPA is a more potent and specific inhibitor of TPH than p-chlorophenylalanine (pCPA). In the present study, pEPA was demonstrated to inhibit competitively and reversibly TPH in vitro (Ki = 32.6 +/- 6.2 microM vs. tryptophan). pEPA displayed little inhibitory activity toward tyrosine hydroxylase (EC 1.14.16.2), the initial and rate-limiting enzyme for catecholamine biosynthesis, and no inhibition of phenylalanine hydroxylase or tyrosinase. In addition, pEPA was a poor ligand for the serotonin transporter and several serotonin receptors.
