Charged Substituents Research Articles

Probing the gas-phase structure of charge-tagged intermediates of a proline catalyzed aldol reaction - vibrational spectroscopy distinguishes oxazolidinone from enamine species.

An l-proline based catalyst with a charged phenyl-pyridium substituent (1) was used to analyze intermediates of an organocatalyzed aldol reaction by infrared multi-photon dissociation (IRMPD) mass spectrometry after transfer into the gas phase via electrospray ionization (ESI). IRMPD spectra were interpreted with the aid of density functional theory (DFT) computations. A structurally restricted enamine species was used as a reference molecule for the calculated vibrational frequencies. A close correlation between theory and experiment was found for the energetically most favoured oxazolidinone structures.

Effects of Charged Ligand Substituents on the Properties of the Formally Copper(III)-Hydroxide ([CuOH]2+) Unit.

With the goal of understanding how distal charge influences the properties and hydrogen atom transfer (HAT) reactivity of the [CuOH]2+ core proposed to be important in oxidation catalysis, the complexes [M]3[SO3LCuOH] (M = [K(18-crown-6)]+ or [K(crypt-222)]+) and [NMe3LCuOH]X (X = BArF4- or ClO4-) were prepared, in which SO3- or NMe3+ substituents occupy the para positions of the flanking aryl rings of the supporting bis(carboxamide)pyridine ligands. Structural and spectroscopic characterization showed that the [CuOH]+ cores in the corresponding complexes were similar, but cyclic voltammetry revealed the E1/2 value for the [CuOH]2+/[CuOH]+ couple to be nearly 0.3 V more oxidizing for the [NMe3LCuOH]2+ than the [SO3LCuOH]- species, with the latter influenced by interactions between the distal -SO3- substituents and K+ or Na+ counterions. Chemical oxidations of the complexes generated the corresponding [CuOH]2+ species as evinced by UV-vis spectroscopy. The rates of HAT reactions of these species with 9,10-dihydroanthracene to yield the corresponding [Cu(OH2)]2+ complexes and anthracene were measured, and the thermodynamics of the processes were evaluated via determination of the bond dissociation enthalpies (BDEs) of the product O-H bonds. The HAT rate for [SO3LCuOH]- was found to be ∼150 times faster than that for [NMe3LCuOH]2+, despite finding approximately the same BDEs for the product O-H bonds. Rationales for these observations and new insights into the roles of supporting ligand attributes on the properties of the [CuOH]2+ unit are presented.

Effect of charged and ortho substituents on 17O NMR chemical shifts of substituted phenyl tosylates in DMSO

AbstractThe 17O nuclear magnetic resonance chemical shifts for 31 ortho‐substituted, para‐substituted, and meta‐substituted phenyl esters of 4‐methylbenzenesulfonic acid (phenyl tosylates, 4‐CH3‐C6H4SO2OC6H4‐X) with the neutral and charged substituents in phenoxy part were measured in DMSO‐d6 at 50°C. The chemical shifts, δ(17O), of the sulfonyl (SO2) and single‐bonded phenoxy (O─Ph) oxygens for para and meta derivatives in the case of neutral substituents showed a good correlation with the Taft σI and σ°R substituent constants. The influence of the ortho substituents on the δ(17O) chemical shifts of the sulfonyl oxygens and single‐bonded phenoxy oxygens was found to be described well with the Charton equation: δ(17O)ortho = δ(17O)H + ρI σI + ρRσ°R + δEBs when the data treatment was performed separately for electron‐donating +R substituents and electron‐withdrawing −R substituents. The substituent constants, σ°X±, for the charged substituents (4‐N+(CH3)3I−, 3‐N+(CH3)3I−, 2‐N+(CH3)3I−, 4‐COO−N+Bu4, 3‐COO−N+Bu4, 2‐COO−N+Bu4) calculated from the δ(17O) chemical shifts for phenyl tosylates in DMSO prove that in DMSO, the compounds involving charged substituents are predominantly in ion pair form and not dissociated into free ions. The same conclusion was drawn recently from the carbonyl IR stretching frequencies, νCO, and the carbonyl carbon 13C chemical shifts for substituted phenyl benzoates, C6H5CO2C6H4‐X, in DMSO. It was shown that for ion pairs of the charged substituents in DMSO and concentrated aqueous salt solution, the same substituent constant, σ°X±, could be used.

More is not always better: finding the right trade-off between affinity and selectivity of a G-quadruplex ligand.

Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.

TADDOL-Derived Cationic Phosphonites: Toward an Effective Enantioselective Synthesis of [6]Helicenes via Au-Catalyzed Alkyne Hydroarylation

A series of cationic phosphonites, all sharing a TADDOL skeleton but decorated with different positively charged substituents at phosphorus, were synthesized and tested as chiral ancillary ligands on the Au-catalyzed intramolecular hydroarylation of appropriate diynes toward carbo[6]helicenes with different substitution patterns. Our studies showed that the Au complexes derived from phosphonites bearing 1,3-dimesityl-1,2,3-triazolium and 1,4-dimesityl-1,2,4-triazolium substituents are the best precatalysts for the desired cyclization in terms of regio- and enantioselectivity of the products obtained. In contrast, all of our attempts to prepare Au complexes from cationic phosphonites derived from CAACs failed, and only ligand decomposition products could be isolated.

Asymmetric Arylation of 2,2,2-Trifluoroacetophenones Catalyzed by Chiral Electrostatically-Enhanced Phosphoric Acids.

A series of highly reactive metal-free chiral phosphoric acids possessing positively charged phosphonium ion substituents are reported and have been applied to Friedel-Crafts alkylations of indoles and 2,2,2-trifluoromethyl aryl ketones. These catalysts are orders-of-magnitude more active and have similar or better enantioselectivities than their noncharged analogues. High tolerance to a range of substrates with electron-withdrawing and electron-donating substituents was also observed.

Profiling Proteasome Activity By Porphyrins

Inspired by some preliminary data and their versatile chemistry we have been investigating the ability of porphyrins to inhibit proteasome activity. Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms. They may bind to the 20S proteasome gates hindering the entrance of the substrate into the catalytic chamber or, by functional connection between the α- and β-rings, allow for allosteric modulation. By contrast, anionic porphyrins may activate the core particle by facilitating the access of the substrate to the proteasome interior, likely regulating the “open”-“closed” allosteric equilibrium. In conclusion, the positioning of the charged substituents represents a sort of key code to fine-tuning porphyrins interference with proteasome gating.

PHOTODYNAMIC INACTIVATION OF PATHOGENIC BACTERIA IN BIOFILMS USING NEW SYNTHETIC BACTERIOCHLORIN DERIVATIVES

Bacteriochlorins as the antimicrobial photosensitizers have a promising future in the face of the unrelenting increase in antimicrobial resistance. The goal of this study was to investigate the infl uence of lipophilicity and number of positively charged substituents in these molecules on the photodynamic inactivation (PDI) of biofi lm bacteria in vitro. Testing how bacteriochlorin derivatives with different properties affect microbes will allow to determine the optimal ratio of these parameters within a single molecule. We have investigated 4 bacteriochlorin derivatives, all of which were synthesized in Organic Intermediates and Dyes Institute. These were: hydrophobic neutral meso-tetra(3-pyridyl) bacteriochlorin (ВС1), amphyphilic tetracationic meso-tetra(1- undecyl-3-pyridyl)bacteriochlorin tetrabromide (ВС2), hydrophilic tetracationic meso-tetra[1-(4'-bromobutyl)-3-pyridyl]bacteriochlorin tetrabromide (ВС3) and octacationic meso-tetra[1-(4'-pyridiniobutyl) -3-pyridyl]bacteriochlorin octabromide (ВС4) . The water-soluble cationic bacteriochlorin derivatives showed the most effective PDI of bacteria in biofi lms. While tetracationic BC3 caused total inactivation of S. aureus 15, octacationic BC4 was bactericidal for P. aeruginosa 32 to the same degree (>99.999%). Interestingly, increasing the number of cationic substituents from 4 to 8 in bacteriochlorin molecules enhances bactericidal action against gram-negative bacteria in biofi lms. The lack of charge-carrying groups and high degree of lipophilicity of PS have negative impact on PDI of biofi lm bacteria. Bacterial membrane damage as a result of PDI can be one of the causes of cell death.

The sequential structure of tripyridiniumylporphyrin pendants in water-soluble copolymers and their association behaviour with tetrasulfonatophenylporphyrin guests: UV-vis absorption and fluorescence emission spectra study.

A novel cationic tripyridiniumylporphyrin monomer, 5-[4-[2-(acryloyloxy)ethoxy]phenyl]-l0,l5,20-tris(N-methyl-4-pyridiniumyl)porphyrinate zinc(ii) (ZnTrMPyP), was synthesized, and its self-aggregation in water was studied by UV-vis absorption. The monomer was copolymerized with acrylamide in water and DMSO, respectively, to prepare the water-soluble polymers P-W and P-D. The aggregation behaviour of the copolymers in aqueous solution was investigated by UV-vis absorption and fluorescence emission spectra. The polymer P-D displayed very similar absorption and emission spectra to those of ZnTrMPyP in water, indicating that the polymer chains in P-D have no significant effect on the aggregate structure of ZnTrMPyP in aqueous media. In comparison, two new absorption bands appeared in the Q band range of polymer P-W and its fluorescence spectra red shifted and the fluorescence quantum yield decreased obviously. These characteristics remained unchanged even in a good solvent for the monomer, suggesting that a new aggregation structure for the porphyrin pendants fixed by the covalent bond was formed. According to the different dispersed states of the porphyrin monomer in water and DMSO, the porphyrin pendants should distribute randomly in the P-D polymer chains while having micro-blocky sequences in polymer P-W. The association behaviour between the copolymers and tetra(p-sulfonatophenyl)porphyrin, TSPP, bearing opposite charged substituents were studied by absorption and emission Spectra and further analyzed by the Benesi–Hildebrand and the Stern–Volmer methods. The results showed that relatively discrete porphyrin pendants in P-D formed a 1 : 1 stoichiometric complex with TSPP and both static and dynamic mechanisms were active in this quenching process, while the tightly associated porphyrin pendants in P-W interacted with TSPP as an entirety and static quenching was dominant in this process. This observation was in accordance with their sequential structure. The polymer P-W has a wider absorption range and higher absorption intensity in the long wavelength region than the porphyrin monomer, which can more efficiently absorb light to accomplish light harvesting in water.

Allosteric mechanism of quinoline inhibitors for HIV RT-associated RNase with MD simulation and dynamics fluctuation network.

The human immunodeficiency virus (HIV) is a retrovirus which infects T lymphocyte of human body and causes immunodeficiency. Reverse transcriptase inhibitors (RTIs) can inhibit some functions of RT, preventing virus synthesis (double-stranded DNA), so that HIV virus replication can be reduced. Experimental results indicate a series of benzimidazole-based inhibitors which target HIV RT-associated RNase to inhibit the reverse transcription of HIV virus. However, the allosteric mechanism is still unclear. Here, molecular dynamics simulations and dynamics fluctuation network analysis were used to reveal the binding mode between the inhibitors and RT-associated RNase. The most active molecule has more hydrophobic and electrostatic interactions than the less active inhibitor. Dynamics correlation network analysis indicates that the most active inhibitor perturbs the network of RT-associated RNase and decreases the correlation of nodes. 3D-QSAR model suggests that two robust and reliable models were constructed and validated by independent test set. 3D-QSAR model also shows that bulky negatively charged or hydrophilic substituent is favorable to bioactivity. These results reveal the allosteric mechanism of quinoline inhibitors and help to improve the bioactivity.

Development of Novel Chemical Probes for Examining Triplet Natural Organic Matter under Solar Illumination.

Excited triplet states of chromophoric dissolved organic matter (3CDOM*) are critical transient species in environmental photochemistry. In the present study, sorbic amine (2,4-hexadien-1-amine) and sorbic alcohol were employed as new probe molecules for triplet measurements and compared to the results measured from sorbic acid under identical conditions. Unlike sorbic acid, sorbic amine and sorbic alcohol were not directly photolyzed under solar irradiation. Photosensitized isomerization of the probes with the conjugated diene structure could yield four geometrical isomers. The separation and quantitative determination of the geometrical isomers were accomplished using HPLC and high-resolution NMR analyses. When photoirradiated Suwannee River natural organic matter (SRNOM) was employed as a source of 3CDOM*, significantly different photosensitized isomerization rates were observed for the diverse charged probes. The bimolecular reaction rate constants between 3SRNOM* and the probes were calculated as (0.42 ± 0.1) × 109 M-1 s-1 for sorbic acid, (1.1 ± 0.1) × 109 M-1 s-1 for sorbic alcohol, and (5.2 ± 0.4) × 109 M-1 s-1 for sorbic amine, respectively. The average apparent Φtriplet was (0.96 ± 0.03)% based on an irradiation range of 290 to 400 nm. We developed highly selective and efficient probes for triplet determination and elucidated the different reaction behaviors of these conjugated dienes containing different charged substituents within the photochemical energy transfer process.

Inhibition of Human Cancer Cell Growth by Analogues of Antimycin A.

In a recent study, several new derivatives of antimycin A (AMA) were produced by means of a novel transacylation reaction, and these were shown to mediate selective toxicity toward cultured A549 human lung epithelial adenocarcinoma cells, as compared with WI-38 normal human lung fibroblasts. The purpose of our study was to investigate whether the analogues all expressed their cytotoxicity by the same mechanism. This was done by studying the effects of the compounds in several types of cell lines. In comparison with 2-O-methylantimycin, which acts at the locus of Bcl-2, none of the new derivatives exhibited a difference in cytotoxicity toward cells expressing different levels of Bcl-2. In cell lines that over- or underexpress estrogen or Her2 receptors, AMA analogue 2 exhibited Her2 receptor dependency at low concentration. Three compounds (1, 4, and 6) exhibited concentration-dependent increases in reactive oxygen species, with 6 being especially potent. Compounds 5 and 6 diminished mitochondrial membrane potential more potently than AMA, and 1 also displayed enhanced activity relative to 2-4. Interestingly, only 1 and AMA displayed strong inhibition of the respiratory chain, as measured by monitoring NADH (reduced nicotinamide adenine dinucleotide) oxidase. Because four of the analogues have positively charged substituents, two of these (4 and 6) were studied to see whether the observed effects were due to much higher level of accumulation within the mitochondria. Their presence in the mitochondria was not dramatically enhanced. Neither of the two presently characterized mechanisms of cell killing by AMA can fully account for the observed results.

Quantum-chemistry descriptors for photosensitizers based on macrocycles.

Phthalocyanines, porphyrins, and chlorins have been widely studied as photosensitizers. Both experimental and computational strategies are employed in order to propose new and more active molecules derived from those macrocycles. In this context, there are two main strategies used: (i) the addition of different substituents and (ii) the complexation of the macrocycle with different metallic ions. In this work, we present selected descriptors based on quantum chemistry calculations for forty macrocycles, including some approved drugs. We have found that density functional theory is a suitable methodology to study the large sets of molecules when applying the B3LYP/LanL2DZ methodology for geometry optimization and TD-OLYP/6-31G(d) for absorption spectrum. The inclusion of solvent effects by means of continuum model is important in order to obtain the accurate electronic data. We have verified that by bonding charged or polar substituents to the macrocycle, it is possible to enhance water solvation as well as to improve spectroscopic properties because molecular orbital contributions for Q band can be affected by some substituents. Selected descriptors, electronic and steric, were pointed out as important to propose the new photosensitizers.

Quinone 1 e- and 2 e-/2 H+ Reduction Potentials: Identification and Analysis of Deviations from Systematic Scaling Relationships.

Quinones participate in diverse electron transfer and proton-coupled electron transfer processes in chemistry and biology. To understand the relationship between these redox processes, an experimental study was carried out to probe the 1e- and 2e-/2H+ reduction potentials of a number of common quinones. The results reveal a non-linear correlation between the 1e- and 2e-/2H+ reduction potentials. This unexpected observation prompted a computational study of 134 different quinones, probing their 1e- reduction potentials, pKa values, and 2e-/2H+ reduction potentials. The density functional theory calculations reveal an approximately linear correlation between these three properties and an effective Hammett constant associated with the quinone substituent(s). However, deviations from this linear scaling relationship are evident for quinones that feature intramolecular hydrogen bonding, halogen substituents, charged substituents, and/or sterically bulky substituents. These results, particularly the different substituent effects on the 1e- versus 2e-/2H+ reduction potentials, have important implications for designing quinones with tailored redox properties.

Aggregation Control of Robust Water-Soluble Zinc(II) Phthalocyanine-Based Photosensitizers.

A water-soluble zinc phthalocyanine (ZnPc) complex with four negatively charged electron-withdrawing sulfonic acid substituents at the nonperipheral positions (α-ZnTSPc) is found to have a high singlet oxygen (1O2) quantum yield and exhibits high photostability. The formation of aggregates is hindered and the highest occupied molecular orbital is significantly stabilized, making α-ZnTSPc potentially suitable for its use as a photosensitizer for photodynamic therapy and photoimmunotherapy. Atomic force microscopy (AFM) reveals that mixtures of the negatively charged α-ZnTSPc complex with a similar positively charged ZnPc were found to result in the self-assembly of one-dimensional accordion-like fibers. Supramolecular fibers can be formed in aqueous solutions through intermolecular electrostatic and donor-acceptor interactions between the two water-soluble ZnPcs.

Effects of neutral and charged substituents on the infrared carbonyl stretching frequencies in phenyl and alkyl benzoates in DMSO

Effects of neutral and charged substituents on the infrared carbonyl stretching frequencies in phenyl and alkyl benzoates in DMSO

Mitochondria-Targeted Analogues of Metformin Exhibit Enhanced Antiproliferative and Radiosensitizing Effects in Pancreatic Cancer Cells.

Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP(+)). In particular, the analogue Mito-Met10, synthesized by attaching TPP(+) to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904-15. ©2016 AACR.

Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.

Highly selective binding of methyl orange dye by cationic water-soluble pillar[5]arenes.

A new water-soluble pillar[5]arene with an amide fragment and triethylammonium groups was synthesized by our original method of aminolysis of the ester groups. Using UV-spectroscopy, it is shown that cationic pillar[5]arenes are able to selectively form 1 : 1 complexes with some hydrophobic anions: the guests with bulky uncharged or negatively charged substituents hindering entry into the macrocycle cavity. Highly selective binding of the most lipophilic guest, methyl orange dye, in the form of organic anion salts by positively charged water-soluble pillar[5]arenes was detected. In the case of the azo dye the appropriate Kass values were 10-100-fold higher than those calculated for the other sulfonic acid derivatives studied. The 2D NMR NOESY (1)H-(1)H spectroscopy confirms the formation of the inclusion complex: negative charge sulfonate head is outside the cavity of pillar[5]arenes and the hydrophobic fragment of the guest is located in the cavity.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1.

Metallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.