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Olefin metathesis has recently emerged as a viable reaction for chemical protein modification. The scope and limitations of olefin metathesis in bioconjugation, however, remain unclear. Herein we report an assessment of various factors that contribute to productive cross-metathesis on protein substrates. Sterics, substrate scope, and linker selection are all considered. It was discovered during this investigation that allyl chalcogenides generally enhance the rate of alkene metathesis reactions. Allyl selenides were found to be exceptionally reactive olefin metathesis substrates, enabling a broad range of protein modifications not previously possible. The principles considered in this report are important not only for expanding the repertoire of bioconjugation but also for the application of olefin metathesis in general synthetic endeavors.

When enzymes are in low dielectric nonaqueous media, it would be expected that their charged groups would be more closely associated with counterions. There is evidence that these counterions may then affect enzymatic activity. Published crystal structures of proteins in organic solvents do not show increased numbers of associated counterions, and this might reflect the difficulty of distinguishing cations like Na(+) from water molecules. In this paper, the placement of several Cs(+) and Cl(-) ions in crystals of the serine protease subtilisin Carlsberg is presented. Ions are more readily identified crystallographically through their anomalous diffraction using softer X-rays. The protein conformation is very similar to that of the enzyme without CsCl in acetonitrile, both for the previously reported ( 1SCB ) and our own newly determined model. No fewer than 11 defined sites for Cs(+) cations and 8 Cl(-) anions are identified around the protein molecule, although most of these have partial occupancy and may represent nonspecific binding sites. Two Cs(+) and two Cl(-) ions are close to the mouth of the active site cleft, where they may affect catalysis. In fact, cross-linked CsCl-treated subtilisin crystals transferred to acetonitrile show catalytic activity several fold higher than the reference crystals containing Na(+). Presoaking with another large cation, choline, also increases the enzyme activity. The active site appears only minimally sterically perturbed by the ion presence around it, so alternative activation mechanisms can be suggested: an electrostatic redistribution and/or a larger hydration sphere that enhances the protein domain.

A phosphine-free palladium catalyst for aqueous Suzuki-Miyaura cross-coupling is presented. The catalyst is active enough to mediate hindered, ortho-substituted biaryl couplings but mild enough for use on peptides and proteins. The Suzuki-Miyaura couplings on protein substrates are the first to proceed in useful conversions. Notably, hydrophobic aryl and vinyl groups can be transferred to the protein surface without the aid of organic solvent since the aryl- and vinylboronic acids used in the coupling are water-soluble as borate salts. The convenience and activity of this catalyst prompts use in both general synthesis and bioconjugation.

Allyl sulfides undergo efficient cross-metathesis in aqueous media with Hoveyda-Grubbs second generation catalyst 1. The high reactivity of allyl sulfides in cross-metathesis was exploited in the first examples of cross-metathesis on a protein surface. S-Allylcysteine was incorporated chemically into the protein, providing the requisite allyl sulfide handle. Preliminary efforts to genetically incorporate S-allylcysteine into proteins are also reported.

The current rule to predict the enantiopreference of subtilisin toward secondary alcohols is based on the size of the substituents at the stereocenter and implies that the active site contains two differently sized pockets for these substituents. Several experiments are inconsistent with the current rule. First, the X-ray structures of subtilisin show there is only one pocket (the S1' pocket) approximately the size of a phenyl group to bind secondary alcohols. Second, the rule often predicts the incorrect enantiomer for reactions in water. To resolve these contradictions, we refine the current rule to show that subtilisin binds only one substituent of a secondary alcohol and leaves the other in solvent. To test this refined empirical rule, we show that the enantioselectivity of a series of secondary alcohols in water varied linearly with the difference in hydrophobicity (log P/P0) of the substituents. This hydrophobicity difference accounts for the solvation of one substituent in water.

Reaction of the hydroxyl group of serine-221 of subtilisin with phenylmethanesulfonylfluoride followed by nucleophilic substitution with sodium hydrogen telluride, a semisynthetic telluroprotein, tellurosubtilisin, was prepared. Tellurosubtilisin, which displays high substrate specificity for aromatic thiols, exhibits remarkable peroxidase activity and catalyzes the reduction of hydrogen peroxide by 3-carboxy-4-nitrobenzenethiol 20 000 times more efficiently than diphenyl diselenide.

Subtilisin Carlsberg-catalyzed hydrolysis of N-chloroacetyl p-toluenesulfinamide favored cleavage of the sulfinamide (S(O)-N) bond with a minor amount ( approximately 25%) of the expected carboxamide (C(O)-N) bond. The sulfinamide hydrolysis was enantioselective (E approximately 17) and yielded remaining starting material enriched in the R-enantiomer and achiral product, sulfinic acid and chloroacetamide, as confirmed by mass spectra and NMR. In contrast, the related subtilisin BPN' and E favored the carboxamide hydrolysis. Hydrolysis of the pseudo-symmetrical N-p-toluoyl p-toluenesulfinamide, which contains a sulfinamide and a carboxamide in similar steric and electronic environments, gave only sulfinamide cleavage (>10:1) for subtilisin Carlsberg, showing that sulfinamide cleavage is the preferred path even when a similar carboxamide is available.

We report the first biocatalytic route to sulfinamides (R-S(O)-NH2), whose sulfur stereocenter makes them important chiral auxiliaries for the asymmetric synthesis of amines. Subtilisin E did not catalyze hydrolysis of N-acetyl or N-butanoyl arylsulfinamides, but did catalyze a highly enantioselective (E > 150 favoring the (R)-enantiomer) hydrolysis of N-chloroacetyl and N-dihydrocinnamoyl arylsulfinamides. Gram-scale resolutions using subtilisin E overexpressed in Bacillus subtilis yielded, after recrystallization, three synthetically useful auxiliaries: (R)-p-toluenesulfinamide (42% yield, 95% ee), (R)-p-chlorobenzenesulfinamide (30% yield, 97% ee), and (R)-2,4,6-trimethylbenzenesulfinamide (30% yield, 99% ee). Molecular modeling suggests that the N-chloroacetyl and N-dihydrocinnamoyl groups mimic a phenylalanine moiety and thus bind the sulfinamide to the active site. Molecular modeling further suggests that enantioselectivity stems from a favorable hydrophobic interaction between the aryl group of the fast-reacting (R)-arylsulfinamide and the S1' leaving group pocket in subtilisin E.

A new procedure for the dynamic kinetic resolution (DKR) of racemic alcohols into single enantiomers is described. This procedure employs surfactant-treated subtilisin as an (S)-selective resolving catalyst and an aminocyclopentadienylruthenium complex as a racemizing catalyst. The DKR is performed best in the presence of an acyl donor such as trifluoroethyl butyrate in THF at room temperature. Eight simple secondary alcohols have been efficiently resolved with high optical purities and good yields. The subtilisin-based DKR is complementary in stereoselectivity to its lipase-based counterpart. For an acyl-carrying alcohol, both subtilisin- and lipase-based DKRs have proceeded equally well to give a pair of enantiomeric products (>99.5% ee each) with opposite optical rotations in high yields (94-95%).

Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

Two enzymes, Mucor javanicus lipase and subtilisin Carlsberg (SC), catalyzed the nonaqueous acylation of doxorubicin (DOX). Compared to the untreated enzyme the rate of DOX acylation at the C-14 position with vinyl butyrate in toluene was 25-fold higher by lipase ion-paired with Aerosol OT (AOT) and 5-fold higher by lipase activated by 98% (w/w) KCl co-lyophilization (3.21 and 0.67 mumol/min g-lipase, respectively, vs 0.13 mumol/min g-lipase). Particulate subtilisin Carlsberg (SC) was nearly incapable of DOX acylation, but ion-paired SC (AOT-SC) catalyzed acylation at a rate of 2.85 mumol/min g-protease. The M. javanicus formulations, AOT-SC, and SC exclusively acylated the C14 primary hydroxyl group of DOX. Co-lyophilization of SC with 98% (w/w) KCl expanded the enzyme's regiospecificity such that KCl-SC additionally acylated the C4' hydroxyl and C3' amine groups. The total rate of DOX conversion with KCl-SC was 56.7 mumol/min g-protease. The altered specificity of KCl-SC is a new property of the enzyme imparted by the salt activation, and represents the first report of unnatural regioselectivity exhibited by a salt-activated enzyme. Using AOT-SC catalysis, four unique selectively acylated DOX analogues were generated, and KCl-SC was used to prepare DOX derivatives acylated at the alternative sites. Cytotoxicities of select derivatives were evaluated against the MCF7 breast cancer cell line (DOX IC50 = 27 nM) and its multidrug-resistant sub-line, MCF7-ADR (DOX IC50 = 27 muM). The novel derivative 14-(2-thiophene acetate) DOX was relatively potent against both cell lines (IC50 of 65 nM and 8 muM, respectively) and the 14-(benzyl carbonate) DOX analogue was as potent as DOX against the MCF7 line (25 nM). Activated biocatalysts and their novel regioselectivity differences thus enabled single-step reaction pathways to an effective collection of doxorubicin analogues.