The growing epidemic of antimicrobial resistance has caused significant morbidity rates worldwide as well as increasing probabilities of cancer recurrence. Efforts to address this epidemic have turned to immunomodulators, a class of molecules which modify the immune system in order to fight off infection. Muramyl dipeptide (MDP) is an immunomodulator of interest due to its specificity for NOD2, a primary receptor involved in immunoregulation. However, use of MDP in vivo causes an overly strong immunostimulatory effect; furthermore, MDP is rapidly cleared from the body due to high solubility and low molecular weight. In order to improve the clinical outlook of MDP, multiple analogues and delivery methods have been explored, with mixed success. MDP analogues have found the most success as adjuvants, although strict FDA approval guidelines limit this approach. Delivery methods for MDP are costly and also induce cytotoxicity, thereby emphasizing the clinical challenges of MDP. Introduced in 1996, Spherical Nucleic Acids (SNAs) are radially oriented oligonucleotides on a nanoparticle surface. SNAs have been shown to readily enter cells with little to no cytotoxicity and in fact, they have found clinical success in a variety of gene delivery and vaccine applications, making them a versatile platform for drug delivery. Herein, we demonstrate that SNAs can be applied as cellular delivery vehicles for MDP. To achieve this, we synthesized dual-layer SNAs whereby two unique oligonucleotides were functionalized onto a gold nanoparticle core. We show that by modulating the surface charge of the SNAs, faster cellular uptake can be achieved, which presents promise for increasing drug delivery and reaching cell compartments of interest. Additionally, we show early evidence that conjugation of MDP to the SNA corona retains MDP activity. Taken together, this work proposes effective SNA designs to deliver muramyl-based immunostimulants, which show promise in the expansion of SNA utility into immunomodulation. / Thesis / Master of Science (MSc) / The growing epidemic of antimicrobial resistance has been linked to deaths, weakened immune systems, and even cancer recurrence. To address this, immunomodulators have become an area of interest. Immunomodulators are capable of modifying the immune system to restore its normal function and fight off infection. Muramyl dipeptide (MDP) is a well-known immunomodulator, however, free MDP is too potent for clinical use. Attempts to modify or deliver MDP have had mixed success. Spherical Nucleic Acids (SNAs) are a 3D orientation of short DNA strands, SNAs are able to readily enter cells with little harmful effects on the cell. Herein, we demonstrate the development of SNAs for MDP delivery. We incorporate two unique DNA strands onto the SNA and leverage charge to enhance cellular uptake. Furthermore, we show early efforts in using the modified SNA as a delivery vehicle for MDP.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A glutamina é o aminoácido livre mais abundante no plasma e músculo, sendo utilizada em elevadas taxas por diversas células na manutenção e promoção de funções essenciais à homeostasia celular. Entretanto, em algumas situações catabólicas, entre as quais exercícios físicos intensos e prolongados ou exaustivos é observada a redução na concentração de glutamina, o que pode comprometer as funções celulares. A suplementação com dipeptídeos de glutamina, tais como a L-alanil-L-glutamina (DIP) pode representar um eficiente meio de fornecimento de glutamina por via oral para o organismo. Desta forma, ratos Wistar machos foram treinados e suplementados com o DIP (1,49 g•kg-1) ou uma solução contendo os aminoácidos L-glutamina (1 g•kg-1) e L-alanina (0,61 g•kg-1) livres (GLN+ALA) ou água (CON), sendo sacrificados em dois tempos, antes (TR) e após (INT) um exercício intenso de natação. No plasma, a concentração de glutamina, glutamato, glicose, amônia, CK, LDH, TNF-α e PgE2 foram avaliadas. No músculo sóleo e gastrocnêmio e no fígado foram determinadas as concentrações de glutamina, glutamato, GSH, GSSG e proteínas totais. Antes do exercício intenso, ambas as suplementações atenuaram a liberação de CK no plasma. O grupo DIP-TR apresentou maior concentração de glutamato, GSH e GSH/GSSG no sóleo e fígado. Maior concentração de glutamina, glutamato, GSH ou GSH/GSSG foi observada no sóleo e fígado do grupo GLN+ALA-TR. Após o exercício intenso, ambas as suplementações atenuaram a liberação de amônia e TNF-α, e no grupo DIP-INT menor concentração de PgE2 foi observada. Maior concentração de glutamina, glutamato e GSH/GSSG no sóleo e glutamato no gastrocnêmio foram encontrados nos grupos DIP-INT e GLN+ALA-INT. No fígado foi encontrada também maior concentração de GSH e GSH/GSSG, decorrentes das suplementações. Os nossos resultados levam à conclusão de que a suplementação oral com L-glutamina e L-alanina na forma livre ou como dipeptídeo, L-alanil-L-glutamina, pode aumentar os estoques musculares e hepáticos de GSH e alterar o estado redox celular, atenuando lesão e a inflamação induzidas pelo exercício físico. / Glutamine is the most abundant free amino acid found in plasma and muscle, and is utilized at high rates by many cells for maintenance and promotion of cell function. However, in some catabolic situations, like intense and prolonged or exhaustive physical exercise, a reduction in GLN availability is observed, fact that may impair the cell functions. Oral supplementation with dipeptides of glutamine, like L-alanyl-L-glutamine may serve as an interesting way to deliver glutamine to the organism. Male Wistar rats were trained and supplemented with DIP (1,49 g•kg-1) or mixture containing free L-glutamine (1 g&#8226kg-1) and Lalanine (0,61 g•kg-1) or water (CON) and sacrificed in distinct times: before (TR) and immediately after (INT) intense exercise of swim. Plasma and serum concentrations of glucose, GLN, glutamate, ammonia, CK, LDH, TNF-• and PgE2 were evaluated. In muscles (soleus and gastrocnemius) and liver GLN, glutamate, GSH, GSSG and total proteins also were evaluated. Low concentration of CK in plasma was observed in both supplemented groups before the exhaustive test. The DIP-TR presented more concentration of glutamate, GSH e GSH/GSSG in the soleus muscle and liver. Higher concentration of glutamine, glutamate and GSH/GSSG was seen in GLN+ALATR group. Both groups supplemented with DIP and GLN+ALA after the exhaustive test, exhibit decreases in ammonia and TNF-α, and the group DIP-INT, demonstrated decrease in PgE2. Glutamine, glutamate and GSH/GSSG increases in the soleus and glutamate in the gastrocnemius increases in groups DIP-INT and GLN+ALA-INT. Increases in GSH and GSH/GSSG in liver were found in the same groups, compared to CON-INT. Our results indicate that oral supplementation with free L-glutamine added to L-alanine or the dipeptide, Lalanyl-L-glutamine, may influence the muscle and liver stores of GSH and the cellular redox state, and this may attenuate damage and inflammation induced by severe physical exercise.

Derivados metálicos de aminoácidos são sistemas modelo simples para o estudo de íons metálicos em estruturas que simulam proteínas. O principal objetivo dessas investigações é avaliar as magnitudes dos parâmetros de acoplamento por interação de troca e relacioná-Ias aos caminhos químicos de supertroca. Foram realizadas medidas através da técnica de Ressonância Paramagética Eletrônica (RPE) em mono cristais dos compostos Cu2+:Gly-Trp e Cu2+:Trp-Gly, nas frequências 9,3 e 33GHz e à temperatura ambiente. Ambos os complexos estão no grupo espacial P212121, com quatro moléculas quimicamente idênticas, mas magneticamente não equivalentes, por cela unitária (Z=4). Essas moléculas formam duas subredes (1 e 2) acopladas por uma interação de troca inter-rede. Cada subrede contem dois íons de cobre não equivalentes, acoplados por uma interação de troca intra-rede. O composto Cu2+:Gly-Trp apresenta duas linhas parcialmente resolvidas em algumas direções do campo magnético aplicado no plano ac, ao passo que há apenas uma linha para todas as orientações nos outros dois planos cristalográficos. O desacoplamento dos espectros leva aos fatores-g moleculares. g⊥=2,0554(3) e g//=2,2086(7) (Cu2+:Gly-Trp). Os espectros de RPE mostram apenas uma linha para todas as direções de campo nos três planos cristalográficos para o Cu2+:Trp-Gly. Portanto, os eixos principais magnéticos são os eixos cristalográficos e a decomposição não é possível através de métodos matemáticos. Medidas utilizando um mono cristal de Zinco (Zn2+:Trp-Gly) ou frequências mais altas (90GHz) podem ajudar na solução desse problema. Os fatores-g moleculares para o composto Cu2+:Trp-Gly foram, então, tomados como sendo aqueles determinados previamente através de medidas em solução aquosa do mesmo composto por M.C.Lara et al. Os dados de RPE juntamente com a teoria de Kubo- Tomita permitem avaliar os acoplamentos intra-rede (J(1)) e inter-rede (J(1,2)) por interação de troca. Os seus valores são /J(1)/I =53,7mK e /J(1,2 /I =7,lmK para o Cu2+:Gly-Trp e /J(1)/=990mK e /J(1,2)/=48,4mK para o Cu2++:Trp-Gly. Eles são discutidos em termos da estrutura cristalina dos compostos, sendo os caminhos para transferência eletrônica constituídos por pontes de hidrogênio e interações cátion-π para o Cu2+:Gly-Trp, e ligações químicas covalentes e interações cátion-π para o Cu2+ +:Trp- Gly. Os valores calculados para /J(1)/ no composto Cu2+: Gly- Trp e /J(1,2)/ no Cu2+:Trp-Gly são uma boa estimativa da magnitude do acoplamento mediado por interações cátion-π. / Metal derivatives of amino acids are simple model systems to study properties of metal ions in protein-like structures. The main purpose of those investigations is to evaluate the magnitudes of exchange coupling parameters and correlate their values with the chemical paths for superexchange. Electron Paramagnetic Resonance (EPR) measurements have been performed in single crystals of the compounds Cu2+:Gly-Trp and Cu2+:Trp-Gly at 9.3 and 33GHz, at room temperature. Both complexes crystallize in the space group P212121, with four chemically identical but magnetically non-equivalent molecules per unit cell (Z=4). These molecules are arranged in two subsets (1 and 2) coupled by an intersubset exchange coupling. Each subset contains two non-equivalent and interacting copper ions (intrasubset exchange coupling). The compound CU2+:Gly-Trp has two partially resolved lines for some field directions in the ac plane, while there is on1y one line along any direction in the other two crystallographic planes. The decoupling of the spectra leads to g⊥=2,0554(3) and g//=2,2086(7) molecular g-factors (Cu2+:Gly-Trp). The EPR spectra show only one line for all field directions in the three crystallographic planes for Cu2+:Trp-Gly. Then, the magnetic principal axes are the crystallographic axes and the g-factor decomposition is not possible by mathematical methods. Measurements of a Zinc (Zn2+:Trp-Gly) single crystal or measurements using high microwave frequencies (90GHz) may help to solve this problem. The molecular g-factors for the compound Cu2+:Trp-Gly were taken to be those ones determined previously from aqueous solution measurements of the same complex by M.C.Lara et al. The EPR data used in conjunction with the Kubo and Tomita\'s theory allow to evaluate the intrasubset (J(1)) e intersubset (J(1,2)) exchange interaction parameters. Their values are /J(1)/ =53,7mK and /J(1,2)/=7,lmK for Cu2+:Gly-Trp and /J(1)/=990mK and /J(1,2)/=48,4mK for Cu2+:Trp-Gly. They have been discussed in terms of the crystal structure of the compounds, and the paths for electron transfer are determined as hydrogen bonds and cation-π interactions for Cu2+:Gly-Trp, and a covalent chemical bond and cation-π interactions for Cu2+:Trp-Gly. The values evaluated for the /J(1)/ in Cu2+:Gly-Trp e /J(1,2)/ in Cu2+:Trp-Gly are a good estimative for the magnitude of the cation-π interactions.

Univ., Diss.--Münster, 2004.

Univ., Diss.--Jena, 1999.

Derivados metálicos de aminoácidos são sistemas modelo simples para o estudo de íons metálicos em estruturas que simulam proteínas. O principal objetivo dessas investigações é avaliar as magnitudes dos parâmetros de acoplamento por interação de troca e relacioná-Ias aos caminhos químicos de supertroca. Foram realizadas medidas através da técnica de Ressonância Paramagética Eletrônica (RPE) em mono cristais dos compostos Cu2+:Gly-Trp e Cu2+:Trp-Gly, nas frequências 9,3 e 33GHz e à temperatura ambiente. Ambos os complexos estão no grupo espacial P212121, com quatro moléculas quimicamente idênticas, mas magneticamente não equivalentes, por cela unitária (Z=4). Essas moléculas formam duas subredes (1 e 2) acopladas por uma interação de troca inter-rede. Cada subrede contem dois íons de cobre não equivalentes, acoplados por uma interação de troca intra-rede. O composto Cu2+:Gly-Trp apresenta duas linhas parcialmente resolvidas em algumas direções do campo magnético aplicado no plano ac, ao passo que há apenas uma linha para todas as orientações nos outros dois planos cristalográficos. O desacoplamento dos espectros leva aos fatores-g moleculares. g⊥=2,0554(3) e g//=2,2086(7) (Cu2+:Gly-Trp). Os espectros de RPE mostram apenas uma linha para todas as direções de campo nos três planos cristalográficos para o Cu2+:Trp-Gly. Portanto, os eixos principais magnéticos são os eixos cristalográficos e a decomposição não é possível através de métodos matemáticos. Medidas utilizando um mono cristal de Zinco (Zn2+:Trp-Gly) ou frequências mais altas (90GHz) podem ajudar na solução desse problema. Os fatores-g moleculares para o composto Cu2+:Trp-Gly foram, então, tomados como sendo aqueles determinados previamente através de medidas em solução aquosa do mesmo composto por M.C.Lara et al. Os dados de RPE juntamente com a teoria de Kubo- Tomita permitem avaliar os acoplamentos intra-rede (J(1)) e inter-rede (J(1,2)) por interação de troca. Os seus valores são /J(1)/I =53,7mK e /J(1,2 /I =7,lmK para o Cu2+:Gly-Trp e /J(1)/=990mK e /J(1,2)/=48,4mK para o Cu2++:Trp-Gly. Eles são discutidos em termos da estrutura cristalina dos compostos, sendo os caminhos para transferência eletrônica constituídos por pontes de hidrogênio e interações cátion-π para o Cu2+:Gly-Trp, e ligações químicas covalentes e interações cátion-π para o Cu2+ +:Trp- Gly. Os valores calculados para /J(1)/ no composto Cu2+: Gly- Trp e /J(1,2)/ no Cu2+:Trp-Gly são uma boa estimativa da magnitude do acoplamento mediado por interações cátion-π. / Metal derivatives of amino acids are simple model systems to study properties of metal ions in protein-like structures. The main purpose of those investigations is to evaluate the magnitudes of exchange coupling parameters and correlate their values with the chemical paths for superexchange. Electron Paramagnetic Resonance (EPR) measurements have been performed in single crystals of the compounds Cu2+:Gly-Trp and Cu2+:Trp-Gly at 9.3 and 33GHz, at room temperature. Both complexes crystallize in the space group P212121, with four chemically identical but magnetically non-equivalent molecules per unit cell (Z=4). These molecules are arranged in two subsets (1 and 2) coupled by an intersubset exchange coupling. Each subset contains two non-equivalent and interacting copper ions (intrasubset exchange coupling). The compound CU2+:Gly-Trp has two partially resolved lines for some field directions in the ac plane, while there is on1y one line along any direction in the other two crystallographic planes. The decoupling of the spectra leads to g⊥=2,0554(3) and g//=2,2086(7) molecular g-factors (Cu2+:Gly-Trp). The EPR spectra show only one line for all field directions in the three crystallographic planes for Cu2+:Trp-Gly. Then, the magnetic principal axes are the crystallographic axes and the g-factor decomposition is not possible by mathematical methods. Measurements of a Zinc (Zn2+:Trp-Gly) single crystal or measurements using high microwave frequencies (90GHz) may help to solve this problem. The molecular g-factors for the compound Cu2+:Trp-Gly were taken to be those ones determined previously from aqueous solution measurements of the same complex by M.C.Lara et al. The EPR data used in conjunction with the Kubo and Tomita\'s theory allow to evaluate the intrasubset (J(1)) e intersubset (J(1,2)) exchange interaction parameters. Their values are /J(1)/ =53,7mK and /J(1,2)/=7,lmK for Cu2+:Gly-Trp and /J(1)/=990mK and /J(1,2)/=48,4mK for Cu2+:Trp-Gly. They have been discussed in terms of the crystal structure of the compounds, and the paths for electron transfer are determined as hydrogen bonds and cation-π interactions for Cu2+:Gly-Trp, and a covalent chemical bond and cation-π interactions for Cu2+:Trp-Gly. The values evaluated for the /J(1)/ in Cu2+:Gly-Trp e /J(1,2)/ in Cu2+:Trp-Gly are a good estimative for the magnitude of the cation-π interactions.

A glutamina é o aminoácido livre mais abundante no plasma e músculo, sendo utilizada em elevadas taxas por diversas células na manutenção e promoção de funções essenciais à homeostasia celular. Entretanto, em algumas situações catabólicas, entre as quais exercícios físicos intensos e prolongados ou exaustivos é observada a redução na concentração de glutamina, o que pode comprometer as funções celulares. A suplementação com dipeptídeos de glutamina, tais como a L-alanil-L-glutamina (DIP) pode representar um eficiente meio de fornecimento de glutamina por via oral para o organismo. Desta forma, ratos Wistar machos foram treinados e suplementados com o DIP (1,49 g•kg-1) ou uma solução contendo os aminoácidos L-glutamina (1 g•kg-1) e L-alanina (0,61 g•kg-1) livres (GLN+ALA) ou água (CON), sendo sacrificados em dois tempos, antes (TR) e após (INT) um exercício intenso de natação. No plasma, a concentração de glutamina, glutamato, glicose, amônia, CK, LDH, TNF-α e PgE2 foram avaliadas. No músculo sóleo e gastrocnêmio e no fígado foram determinadas as concentrações de glutamina, glutamato, GSH, GSSG e proteínas totais. Antes do exercício intenso, ambas as suplementações atenuaram a liberação de CK no plasma. O grupo DIP-TR apresentou maior concentração de glutamato, GSH e GSH/GSSG no sóleo e fígado. Maior concentração de glutamina, glutamato, GSH ou GSH/GSSG foi observada no sóleo e fígado do grupo GLN+ALA-TR. Após o exercício intenso, ambas as suplementações atenuaram a liberação de amônia e TNF-α, e no grupo DIP-INT menor concentração de PgE2 foi observada. Maior concentração de glutamina, glutamato e GSH/GSSG no sóleo e glutamato no gastrocnêmio foram encontrados nos grupos DIP-INT e GLN+ALA-INT. No fígado foi encontrada também maior concentração de GSH e GSH/GSSG, decorrentes das suplementações. Os nossos resultados levam à conclusão de que a suplementação oral com L-glutamina e L-alanina na forma livre ou como dipeptídeo, L-alanil-L-glutamina, pode aumentar os estoques musculares e hepáticos de GSH e alterar o estado redox celular, atenuando lesão e a inflamação induzidas pelo exercício físico. / Glutamine is the most abundant free amino acid found in plasma and muscle, and is utilized at high rates by many cells for maintenance and promotion of cell function. However, in some catabolic situations, like intense and prolonged or exhaustive physical exercise, a reduction in GLN availability is observed, fact that may impair the cell functions. Oral supplementation with dipeptides of glutamine, like L-alanyl-L-glutamine may serve as an interesting way to deliver glutamine to the organism. Male Wistar rats were trained and supplemented with DIP (1,49 g•kg-1) or mixture containing free L-glutamine (1 g&#8226kg-1) and Lalanine (0,61 g•kg-1) or water (CON) and sacrificed in distinct times: before (TR) and immediately after (INT) intense exercise of swim. Plasma and serum concentrations of glucose, GLN, glutamate, ammonia, CK, LDH, TNF-• and PgE2 were evaluated. In muscles (soleus and gastrocnemius) and liver GLN, glutamate, GSH, GSSG and total proteins also were evaluated. Low concentration of CK in plasma was observed in both supplemented groups before the exhaustive test. The DIP-TR presented more concentration of glutamate, GSH e GSH/GSSG in the soleus muscle and liver. Higher concentration of glutamine, glutamate and GSH/GSSG was seen in GLN+ALATR group. Both groups supplemented with DIP and GLN+ALA after the exhaustive test, exhibit decreases in ammonia and TNF-α, and the group DIP-INT, demonstrated decrease in PgE2. Glutamine, glutamate and GSH/GSSG increases in the soleus and glutamate in the gastrocnemius increases in groups DIP-INT and GLN+ALA-INT. Increases in GSH and GSH/GSSG in liver were found in the same groups, compared to CON-INT. Our results indicate that oral supplementation with free L-glutamine added to L-alanine or the dipeptide, Lalanyl-L-glutamine, may influence the muscle and liver stores of GSH and the cellular redox state, and this may attenuate damage and inflammation induced by severe physical exercise.

Currently aquaculture is the largest growing food sector in the world, however, its future growth is limited by its heavy reliance on fishmeal (FM). Overfishing of wild marine fish stocks used for FM is putting too much pressure on the stocks, making FM unsustainable as a dominant protein source as aquaculture continues to grow. Plant proteins (PP) are an ideal alternative to FM because of their wide availability and relatively low cost. Soybean meal (SBM) is the most commonly used PP in aquaculture, but its inclusion in feeds is limited due to reduced digestibility and the presence of antinutritional factors ultimately leading to a reduced growth performance of fish that are fed with the SBM-based diet. Therefore, the goal of this thesis was to mitigate the negative effects of PP and improve the growth of fish fed with a PP-based diet, utilizing SBM as our PP in feeds. Three feeding trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish. The first trial (Chapter 2), tested the effect of nutritional programming (NP) on the utilization of SBM in zebrafish (Danio rerio). NP is the theory that an organism can be ‘programmed’ to better utilize a dietary component by being exposed to that component in its early life stages. This study also tested the effect of NP through the broodstock by exposing the broodstock to SBM for 4 weeks prior to breeding. In addition, a combination of both programming techniques was also tested. The results found that neither of the programming techniques had a significant effect on the growth performance of the zebrafish. Among the two groups from the programmed broodstock, the group that also received early stage NP had a significantly higher expression of PepT1, a di- and tri- peptide transporter. Also, the dual programmed group had the highest length-to-width ratio of the distal villi among groups that were fed SBM, which signifies an increase in surface area for nutrient absorption in the intestine. The findings from this study suggest that early stage NP may increase the absorption of nutrient from PP-based feeds within the intestine. The second feeding trial (Chapter 3), utilized the supplementation of health-promoting dipeptides to improve the use of SBM-based feeds for zebrafish. The three dipeptides used in this study were alanyl-glutamine, carnosine, and anserine. The five groups in this study consisted of three groups receiving an SBM-based diet with one of the dipeptides supplemented into it, a (-) Control group receiving a non-supplemented SBM-based diet, and a (+) Control group receiving a FM-based diet. Both the alanyl-glutamine and carnosine supplemented groups experienced a significantly higher weight gain compared to the (-) Control group. In addition, the alanyl-glutamine supplemented group had a significantly higher length-to-width ratio of the intestinal villi and, had a numerically higher expression of both nutrient absorption genes measured, PepT1 and fabp2, compared to the (-) Control group. This finding suggests that the supplementation of alanyl-glutamine into SBM-based diets may improve the intestinal absorptive capacity of the fish fed with the SBM. The results from this study also support the use of both alanyl-glutamine and carnosine supplementation as a means of improving the growth performance of fish fed with a SBM-based diet. The third feeding trial (Chapter 4), was conducted on largemouth bass (LMB) (Micropterus salmoides). This study also focused on NP as a means of improving the utilization of dietary SBM, similar to Chapter 2. In this study, live feed was used as a vector to program the larval LMB to SBM. The programmed group in this study received Artemia nauplii that were enriched with a SBM solution, during the larval stage. The bass were then fed with a FM-diet for 7 weeks, before being reintroduced to SBM for the final 6 weeks of the study (PP-Challenge). The programmed LMB experienced a significantly higher weight gain compared to the non-programmed fish also undergoing the PP-Challenge, and achieved a weight gain similar to that of the LMB that were being fed with an FM-diet. In addition, the programmed LMB had significantly longer distal villi and a higher length-to-width ratio of the villi, compared to the non-programmed group. The findings from this study support the use of live feed as a vector for NP and improving the growth performance of a carnivorous aquaculture species fed with a SBM-based diet. The overall findings from these studies suggest that both NP and dipeptide supplementation are feasible means of improving the utilization of SBM in fish. The mechanism behind NP seems to lie in the intestine. In both zebrafish and LMB, NP was found to reduce the inflammatory impact on the intestine and increase the surface area for absorption of the intestinal villi. The supplementation of alanyl-glutamine had similar effects on the intestine as NP and improved the growth performance of zebrafish. The observations from these studies seem to point to mitigating the negative effects of SBM on the intestine as the key to improving the growth performance of fish fed with a SBM-based diet.

The design, synthesis and evaluation of chiral selectors that allow the determination of enantiomeric composition using electrospray ionization mass spectrometry are detailed herein. The enantiomers of the chiral selector were mass labeled at a distant site from the chiral recognition sites of the molecules. The mass-labeled enantiomers were mixed in a one-to-one ratio to form a quasi-racemate. Chiral recognition can be observed by comparing relative abundances of the pseudo-diastereomeric selector-analyte complexes in the mass spectrum. The observed sense of chiral recognition with mass spectrometry was consistent with that observed chromatographically using a corresponding chiral stationary phase in every case. The complex intensity fraction (CIF, intensity of one selector-analyte complex divided by the sum of the intensities for both selector-analyte complexes) is linear with the enantiomeric composition. The slope of this line is an indication of the extent of the enantioselectivity: the larger the slope, the more significant the enantioselectivity. In addition, this line can be used as a calibration curve for the quantitative determination of enantiomeric composition of the same analyte with unkown enantiomeric composition. Amide derivatives of DNB-amino acids were first used as pseudo-enantiomeric chiral selectors in the presence of added lithium chloride. The enantioselectivity values were smaller than those observed on chiral HPLC using the corresponding chiral stationary phase. The use of deprotonated DNB-amino acids as chiral selectors provides higher enantioselectivities, but with low ion abundances. Tertiary amine appended analogues of the chiral stationary phase DNB-Leucine were prepared. The amine was appended to provide a site for ready ionization (through protonation). The performance of chiral selectors of this type was compared to the original chiral selectors that lack this functional group. Chiral recognition was also observed in a reciprocal sense using proline-derived pseudo-enantiomeric chiral selectors and analytes similar to DNB-amino acid esters or amides. Optimization of the electrospray ionization conditions provided similar enantioselectivities to those from chiral HPLC. Libraries of tertiary amine appended derivatives of DNB-dipeptides, which were prepared through combinatorial peptide synthesis, were screened using electrospray ionization mass spectrometry. The use of electrospray ionization mass spectrometry as a discovery tool for new chiral selectors is discussed.