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Abstract(s)
A cromatografia de afinidade (AC) baseada em aminoácidos apresenta-se como uma abordagem promissora para purificar DNA de plasmídico (pDNA), uma vez que combina a seletividade de uma interacção que ocorre naturalmente, com a simplicidade de um ligando de baixo peso molecular. Este estudo irá concentrar-se na síntese química de dipeptídeos L-triptofano-L-triptofano e L-triptofano-L-arginina, seguida da sua imobilização na matriz sefarose previamente ativada para explorar a possibilidade de utilizar estes suportes na purificação plasmídico do pDNA.Também o aminoácido L-triptofano e o suporte sefarose-L-triptofano foram estudados como estratégia de ligando de afinidade. A síntese química dos dipeptídeos L-triptofano-L-triptofano e L-triptofano-L-arginina foi realizada através da condensação do dicarbonato de di-terc-butila (BOC) com L-triptofano metil éster ou L-arginina metil éster na presença de 1,3-diciclohexilcarbodiimida (DCC). Os grupos metilo e BOC foram desprotegidos com hidróxido de sódio e ácido trifluoroacético, respetivamente. As interacções específicas entre os suportes sintetizados sefarose L-triptofano, sefarose L-triptofano-L-triptofano e sefarose L-triptofano-L-arginina e os 5'-mononucleotideos foram estudados por diferença de transferência de saturação (STD) - ressonância magnética nuclear (RMN). As constantes de dissociação de equilíbrio entre os ligandos L-triptofano, L-triptofano-L-triptofano e L-triptofano-L-arginina e as isoformas superenrolada (sc), circular aberta (oc) e linear(ln) do pVax-Lacz foram determinadas por ressonância de plasmónica de superfície (RPS) utilizando o biosensor ótico Biacore T200 equipado com um sensor chip CM5. A imobilização dos ligandos L-triptofano, L-triptofano-L-triptofano e L-triptofano-L-arginina no sensor chip CM5 foi efetuada utilizando o método de acoplamento do grupo amina. Por RPS, verifica-se que apenas o ligando L-triptofano-L-triptofano em Tris-HCl tem sinal de RPS com a isoforma circular aberta apresentando afinidade alta (KD= 3,04x10-9 M). Enquanto que o L-triptofano-L-arginina tem maior afinidade em Hepes a T=10°C com a isoforma linear com KD= 2,51x10-9M a isoforma sc é a que apresenta a menor afinidade (10-7 M) com os ligandos a ambas as temperaturas, nomeadamente com o L-triptofano-L-arginina. Por RMN-STD verifica-se que o 5’-TMP interage com os suportes pelo grupo metilo, enquanto que o 5´-AMP interage com os dipeptídeos pela desoxirribose.O 5’-GMP e o 5’-CMP interagem preferencialmente com L-triptofano e L-triptofano-L-triptofano pelos protoes adajcentes ao grupo fosfato.
Desta modo, a utilização das técnicas STD-RMN e SPR permitiu fazer o screening das interações dos ligandos-5´mononucleótidos e determinar a afinidade do ligando:plasmideo para a purificação do pDNA em AC.
Amino acid-based affinity chromatography appears as a promising approach to purify plasmid DNA since it combines the selectivity of a naturally occurring interaction with the simplicity of a single small ligand. This study will focus on the chemical synthesis of the dipeptides L-tryptophan-L-tryptophan and L-tryptophan-L-arginine, follow by their immobilization on activated Sepharose to explore the possibility of using these supports in plasmid purification. The amino acid L-tryptophan and their support are also studied as affinity ligand. The synthesis of the L-tryptophan-tryptophan and L-tryptophan-arginine were performed by condensation of N-tertbutoxycarbonyl (BOC) tryptophan with L- tryptophan or L-arginine methyl esters in the presence of 1,3-dicyclohexylcarbodiimide (DCC). The deprotection of methyl group and BOC is done with sodium hydroxide and trifluoracetic acid, respectively. The specific interactions between the supports L-tryptophan, L-tryptophan-tryptophan and L-tryptophan-arginine, previously synthetized, and 5’-mononucleotides are accomplished by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. The binding affinity of L-tryptophan, L-tryptophan-L-tryptophan and L-tryptophan-L-arginine towards supercoiled (sc), open-circular (oc) and linear (ln) isoforms of pVax-Lacz is determined by measuring the equilibrium dissociation constants (KD) by surface plasmon resonance (SPR) using the biosensor T200 equipped with a CM5 sensor chip. The immobilization of L-tryptophan, L-tryptophan-L-tryptophan and L-tryptophan-L-arginine on CM5 surface was performed using the amine-coupling method. From SPR, in Tris-HCl only signals are finding to open-circular and the highest affinity is with L-tryptophan-L-tryptophan (KD=3,04x10-9M) at T=10°C. While in Hepes, the L-tryptophan-L-arginine has the highest affinity with linear isoform (KD= 2,51x10-9M) at T=10°C. The sc isoform show the lowest affinity (10-7M) with all ligands surface at both temperatures, namely with L-tryptophan-L-arginine. From STD-NMR, 5’-TMP interact namely through the metyl group of tymine, while 5´-AMP interact with the dipeptides through the deoxyribose. The 5’-GMP and 5’-CMP interact with L-tryptophan and L-tryptophan-L-tryptophan namely through protons adajcent to phosphat group. Thus, STD-NMR and SPR techniques allow us to screen the interactions of supports-5´mononucleotides and determine the binding affinity of these ligands to plasmid isoforms for application in AC.
Amino acid-based affinity chromatography appears as a promising approach to purify plasmid DNA since it combines the selectivity of a naturally occurring interaction with the simplicity of a single small ligand. This study will focus on the chemical synthesis of the dipeptides L-tryptophan-L-tryptophan and L-tryptophan-L-arginine, follow by their immobilization on activated Sepharose to explore the possibility of using these supports in plasmid purification. The amino acid L-tryptophan and their support are also studied as affinity ligand. The synthesis of the L-tryptophan-tryptophan and L-tryptophan-arginine were performed by condensation of N-tertbutoxycarbonyl (BOC) tryptophan with L- tryptophan or L-arginine methyl esters in the presence of 1,3-dicyclohexylcarbodiimide (DCC). The deprotection of methyl group and BOC is done with sodium hydroxide and trifluoracetic acid, respectively. The specific interactions between the supports L-tryptophan, L-tryptophan-tryptophan and L-tryptophan-arginine, previously synthetized, and 5’-mononucleotides are accomplished by saturation transfer difference (STD)-nuclear magnetic resonance (NMR) spectroscopy. The binding affinity of L-tryptophan, L-tryptophan-L-tryptophan and L-tryptophan-L-arginine towards supercoiled (sc), open-circular (oc) and linear (ln) isoforms of pVax-Lacz is determined by measuring the equilibrium dissociation constants (KD) by surface plasmon resonance (SPR) using the biosensor T200 equipped with a CM5 sensor chip. The immobilization of L-tryptophan, L-tryptophan-L-tryptophan and L-tryptophan-L-arginine on CM5 surface was performed using the amine-coupling method. From SPR, in Tris-HCl only signals are finding to open-circular and the highest affinity is with L-tryptophan-L-tryptophan (KD=3,04x10-9M) at T=10°C. While in Hepes, the L-tryptophan-L-arginine has the highest affinity with linear isoform (KD= 2,51x10-9M) at T=10°C. The sc isoform show the lowest affinity (10-7M) with all ligands surface at both temperatures, namely with L-tryptophan-L-arginine. From STD-NMR, 5’-TMP interact namely through the metyl group of tymine, while 5´-AMP interact with the dipeptides through the deoxyribose. The 5’-GMP and 5’-CMP interact with L-tryptophan and L-tryptophan-L-tryptophan namely through protons adajcent to phosphat group. Thus, STD-NMR and SPR techniques allow us to screen the interactions of supports-5´mononucleotides and determine the binding affinity of these ligands to plasmid isoforms for application in AC.
Description
Keywords
Dipeptídeos Dna Plasmídico L-Triptofano Ressonância Magnética Nuclear Ressonância Plasmónica de Superfície