Catecholamine Derivatives as Novel Crosslinkers for the Synthesis of Versatile Biopolymers.

Catecholamine metabolites are not only involved in primary metabolism, but also in secondary metabolism, serving a diverse array of physiologically and biochemically important functions. Melanin, which originates from dopa and dopamine, found in the hair, eye, and skin of all animals, is an important biopolymeric pigment. It provides protection against damaging solar radiation to animals. N-Acetyldopamine and N-ß-alanyldopamine play a crucial role in the hardening of the exoskeletons of all insects. In addition, insects and other arthropods utilize the melanogenic process as a key component of their defense systems. Many marine organisms utilize dopyl peptides and proteins as bonding materials to adhere to various substrata. Moreover, the complex dopa derivatives that are precursors to the formation of the exoskeletons of numerous marine organisms also exhibit antibiotic properties. The biochemistry and mechanistic transformations of different catecholamine derivatives to produce various biomaterials with antioxidant, antibiotic, crosslinking, and gluing capabilities are highlighted. These reactivities are exhibited through the transient and highly reactive quinones, quinone methides, and quinone methide imine amide intermediates, as well as chelation to metal ions. A careful consideration of the reactivities summarized in this review will inspire numerous strategies for synthesizing novel biomaterials for future medical and industrial use.

The impact of the complexing agent on the sensitivity of collision-induced dissociation spectra to fatty acid position for a set of XYZ-type triglycerides.

RATIONALE: The development of an automated platform for the positional analysis of triglycerides (TAGs) based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) continues to be pursued. This work evaluates the positional sensitivities of the collision-induced dissociation (CID) spectra of a representative set of XYZ triglycerides using sodium, lithium, and ammonium salts as complexing agents. METHODS: A set of triglycerides were synthesized and analyzed via ESI-MS/MS using an ion trap mass spectrometer. Using three different complexing agents, the product ion spectra of the corresponding precursor ions for twelve XYZ TAGs were collected, where X, Y, and Z represent C16:0 , C18:1(c-9) , C18:2(cc-9,12) , and C20:4(cccc-5,8,11,14) fatty acid chains. These data were then used to prepare ternary plots for four positional isomer systems to evaluate the positional sensitivity differences among the three different complexing agents. RESULTS: The positional sensitivities for each of the four positional isomer systems were robust for the sodium and lithium adducts. The CID data for the sodium and lithium TAGs demonstrated an unfavorable loss of the fatty acid in the center position and showed a higher sensitivity to fatty acid position, when compared with the CID data for ammonium adducts, especially for the arachidonic acid containing triglycerides. CONCLUSIONS: The data shows that that the relative abundances of the DAG product ions for the XYZ-type TAGs when using sodium and lithium complexing agent adducts are sensitive to fatty acid position and are consistent for the diverse array of TAGs studied in this work. This suggests that using sodium or lithium as the complexing agent may be advantageous for the development of an automated platform for the positional analysis of complex TAG mixtures based on ESI-MS/MS.

Protease Degradation of Protein Coronas and Its Impact on Cancer Cells and Drug Payload Release.

The effect of matrix metalloproteinases (MMPs) on preformed protein coronas around spherical gold nanoparticles (AuNPs) was studied. Protein coronas of different compositions (human serum, human serum albumin, and collagen IV) were formed around AuNPs and characterized. The protease MMP-9 had different effects on the corona depending on the corona composition, resulting in different changes to the corona hydrodynamic diameter ( DH). When incubated with PANC-1 cells, the corona showed evidence of both increases as well as decreases in DH. Varying the composition of the corona influenced the MMP-9 activity. Furthermore, the corona was influenced not only by the protease activity of the MMP-9 but also by its ability to exchange with proteins in the preformed corona. This exchange could also occur with proteins in the media. Thus, the net effect of the MMP-9 was a combination of the MMP-9 protease activity and also exchange. Time scales for the exchange varied depending on the nature that make up the protein corona (weakly vs strongly bound corona proteins). Mass spectrometry was used to probe the protein corona composition and supported the exchange and degradation model. Together, these results indicate that the mechanism of protease activity on AuNP coronas involves both rearrangement and exchange, followed by degradation.

Modeling the fragmentation patterns of triacylglycerides in mass spectrometry allows the quantification of the regioisomers with a minimal number of standards.

Mass spectrometry allows the relative quantification of the regioisomers of triacylglycerides by the calibration of their fragmentation patterns. However, due to the plethora of regioisomers of triacylglycerides, calibration with every standard is not feasible. An analytical challenge in the field is the prediction of the fragmentation patterns of triacylglycerides to quantify their regioisomers. Thus, we aimed to model these fragmentation patterns to quantify the regioisomeric composition, even for those without commercially available standards. In a first step, we modeled the fragmentation patterns of the regioisomers of triacylglycerides obtained from different published datasets. We found the same qualitative trends of fragmentation beyond differences in the type of adduct in these datasets (both [M+NH4]+ and [M+H]+), and the type of instrument (orbitrap, Q-ToF, ion-trap, single quadrupole, and triple quadrupole). However, the quantitative trends of fragmentation were adduct and instrument specific. From these observations, we modeled quantitatively the common trends of fragmentation of triacylglycerides in every dataset. In a second step, we applied this methodology on a Synapt G2S Q-ToF to quantify the regioisomers of triacylglycerides in sunflower and olive oils. The results of our quantification were in good agreement with previous published quantifications of triacylglycerides, even for regioisomers that were not present in the training dataset. The species with more than two highly unsaturated fatty acids (arachidonic, eicosapentaenoic, and docosahexaenoic acids) showed a complex behavior and lower predictability of their fragmentation patterns. However, this framework presents the capacity to model this behavior when more data are available. It would be also applicable to standardize the quantification of the regioisomers of triacylglycerides in an inter-laboratory ring study.

The Impact of the Complexing Cation on the Sensitivity of the Collisional-Induced Dissociation Spectra to Fatty Acid Position for a Set of YXY/YYX-type Triglycerides.

RATIONAL: The development of an automated platform for the positional analysis of triglycerides based on electrospray tandem mass spectrometry continues to be pursued. This work compares the positional sensitivities of the collisional-induced dissociation spectra for a representative set of YXY/YYX triglycerides using ammonium, silver, sodium and lithium as complexing agents. METHODS: A set of triglycerides were synthesized and analyzed by electrospray tandem mass spectrometry using an ion trap mass spectrometer. Using different salt additives, the product ion spectra of the corresponding parent ions for twelve systems of the form YXY/YYX, where Y and X represent C16:0 , C18:1(c-9), C18:2(cc-9,12) and C20:4(cccc-5,8,11,14) , were collected. The data was used to prepare two-point calibration plots for each of the twelve positional isomer systems using each of the four complexing agents. RESULTS: The positional sensitivities for all twelve positional isomer systems were robust for both the sodium and lithium TAG adducts. The CID data for both the sodium and lithium TAG adducts are much less sensitive to the degree of unsaturation and double bond position of the fatty acids constituents than the CID data for the ammonium adducts. CONCLUSION: Using sodium or lithium TAG adducts may be advantageous for the development of an accurate predictive model for performing positional analysis of complex TAG mixtures based on electrospray tandem mass spectrometry. Ammonium adducts are likely complicated by the ability of the ammonium ion to provide extra stability to some parent ions through hydrogen bond-like interactions.

A simple and economical strategy for obtaining calibration plots for relative quantification of positional isomers of YYX/YXY triglycerides using high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Positional analysis of intact triglycerides could provide greater insights into the link between fatty acid position and lipotoxic diseases. However, this methodology has been impeded by lack of commercial availability of positionally pure triglycerides. This work reports on a strategy for defining calibration plots for YXY/YYX triglyceride systems based on the product ion intensities in the collision-induced dissociation spectra of ammoniated precursor ions. METHODS: A set of triglycerides were synthesized and analyzed by electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer. The product ion spectra of the ammoniated precursor ions were collected for 42 triglyceride systems of the form YXY/YYX, where Y represents C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) . Three-point calibration plots were prepared by plotting the relative abundance of the YY+ product ion vs. the relative abundance of the YYX positional isomer. RESULTS: The calibration plots were shown to give relative abundances of positional isomers accurate to within ±0.02 for most systems. Using an ion trap, under a controlled set of collision parameters, the slopes of the calibration plots can be used to compare the sensitivities of the product ion intensities to fatty acid position for various triglyceride systems. The average slopes of the calibration plots for the C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) systems were 0.29 ± 0.05, 0.21 ± 0.05 and 0.045 ± 0.005, respectively. CONCLUSIONS: While the presence of multiple unsaturated fatty acids tends to slightly decrease the slopes of the calibration plots, the data suggest that the sensitivities are sufficient for performing positional analysis of most triglyceride systems. However, the presence of unsaturated fatty acids that contain double bonds close to the carbonyl group, such as arachidonic acid, tends to dramatically decrease positional sensitivity.

Mass spectrometric studies shed light on unusual oxidative transformations of 1,2-dehydro-N-acetyldopa.

RATIONALE: Lamellarins are a group of over 70 plus bioactive marine natural compounds possessing a 6,7-dihydroxycoumarin moiety. Although they appear to derive from 3,4-dihydroxyphenylalanine (dopa), practically nothing is known about the metabolic fate of these compounds. Biochemical considerations indicate that they could arise from a N-acetyl-1,2-dehydrodopa precursor through oxidative cyclization reaction. METHODS: To assess the above hypothesis, we synthesized N-acetyl-1,2-dehydrodopa and conducted oxidation studies with commercially available mushroom tyrosinase and evaluated the course of the reaction with reversed-phase liquid chromatography/mass spectrometry (LC/MS). RESULTS: Mushroom tyrosinase readily oxidized N-acetyl-1,2-dehydrodopa - not to the normally expected quinone - but to an unstable quinone methide isomer, which rapidly cyclized to produce the dihydroxycoumarin product, 3-aminoacetyl esculetin. Interestingly, 3-aminoacetyl esculetin was further oxidized to a second quinone methide derivative that exhibited an addition reaction with the parent dihydroxycoumarin generating dimeric and other oligomeric products in the reaction mixture. CONCLUSIONS: LC/MS analysis of the N-acetyl-1,2-dehydrodopa oxidation reaction reveals not only a possible novel oxidative cyclization route for the biosynthesis of coumarin-type dehydrodopa compounds in marine organisms, but also unusual oxidative transformations of dehydro dopa derivatives.

Examining the collision-induced decomposition spectra of ammoniated triglycerides. III. The linoleate and arachidonate series.

A series of positionally pure triglycerides (TAGs) of the form LXL, YLY, AXA, and YAY was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. L and A represent the linoleate and arachidate moieties, respectively, and X and Y represent large arrays of fatty acid moieties of various chain lengths, degree of unsaturations, double-bond positions, and cis/trans configurations. The abundances of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of these parameters. The major CID products, the diglyceride (DAG) product ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. The following trends are observed in the data. TAGs with higher degrees of unsaturation tend to show greater relative abundances of MH(+) in the CID spectra of their ammoniated precursor ions. The position of the fatty acid constituents along the glycerol backbone also seems to influence the abundances of the MH(+) ion in the CID spectra of the ammoniated precursor ions. A fatty acid constituent with double bonds along the fatty acid chain positioned close to the carbonyl promotes the formation of the DAG product ion that corresponds to its loss upon CID of the ammoniated precursor ion. Linoleic acid substituents also seem to promote the formation of DAG product ions, but to a lesser extent. Data for the YAY TAGs are used to predict the abundances of the product ions in the CID spectra of ammoniated YAX TAGs. These data are discussed in context of a broader project to develop and validate software algorithims to support a platform for comprehensive analysis of complex mixtures of TAGs.

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. II. The PXP/YPY series.

A series of positionally pure triglycerides (TAGs) of the form PXP and YPY, where P is the palmitate moiety and X and Y are large arrays of different fatty acid moieties, is synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride (DAG) fragment ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. Different trends for each of these series are observed. Trends in the intensities of these fragment ions are also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional intensities of MH(+) increase with increasing degree of unsaturation in the TAGs. MH(+) is absent in the CID spectra of triglycerides containing three saturated fatty acid moieties, suggesting that the presence of double bonds fosters the formation of MH(+). Double bonds positioned close to the carbonyl carbon along the fatty acid chain promote the formation of the DAG fragment ion corresponding to the loss of the fatty acid. The collection of PXP/YPY data produced in this work is used to test the mechanisms of the formation and decomposition of ammoniated TAGs that were previously presented. The YPY data are used to predict the intensities of the fragment ions in the CID spectra of YPX-type TAGs. The limitations of the mathematical approach used in these predictions are discussed in context of a broader plan to develop a software platform for comprehensive analysis of complex TAG mixtures.

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. I. The OXO/YOY series.

A series of positionally pure triglycerides (TAGs) of the form OXO and YOY, where O is the oleate moiety and X and Y are large arrays of different fatty acid moieties, was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs (ammonium ion adducts) were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride fragment ions and the MH+ ion, were plotted as functions of chain length for the saturated and mono-unsaturated series of X and Y. Different trends for each of these series were observed. Trends in the abundances of these fragment ions were also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional abundances of the MH+ ions vary linearly with degree of unsaturation. However, the presence of double bonds positioned close to the carbonyl carbon of the fatty acid chain promotes the formation of the diglyceride fragment ion corresponding to loss of that fatty acid. Mechanisms of the formation and decomposition of ammoniated TAGs are proposed that fit the trends observed in the data. Extensions of this work are described, and a vision of a derived library of CID spectra is discussed as a platform for comprehensive analysis of complex TAG mixtures.

Determining the relative amounts of positional isomers in complex mixtures of triglycerides using reversed-phase high-performance liquid chromatography-tandem mass spectrometry.

A reversed-phase HPLC-tandem mass spectrometry (RP-HPLC-MS-MS) method was refined for the positional analysis of complex mixtures of TAG. This method has the advantages of speed, ease of automation, and specificity over traditional digestion-based methods for the positional analysis of TAG. Collision-induced dissociation (CID) of ammoniated TAG in an ion-trap mass spectrometer produced spectra that were dependent on the FA position. Dominant DAG fragments were formed from the loss of a FA moiety from the ammoniated TAG species. The loss of FA in the outer positions was favored over their loss in the central position. The combination of RP-HPLC and CID produced spectra that were free of the isotope effects that can complicate spectral interpretation in existing methods. The combination also provided selectivity based on the chromatographic fractionation of TAG, in addition to the selectivity inherent in the CID process. Proof-of-concept experiments were performed with binary mixtures of TAG from the SOS/SSO, OSO/OOS, and the PSO/POS/SPO positional isomer systems (where S is 18:0, stearic acid; O is 18:1 (cis-9), oleic acid; and P is 16:0, palmitic acid). Plots of fractional DAG fragment intensities vs. fractional composition of the binary mixtures were linear. These plots were used to determine the fractional composition of each of these isomeric systems in a variety of vegetable oils and animal fats. Current limitations, future developments, and applications of this method are discussed.