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Isabella I of Castile (22 April 1451 – 26 November 1504) was a Queen of Castile and León, and the wife of King Ferdinand II. Furthermore, she was a very religious person and often had contact with Tomás de Torquemada, Inquisitor General and her personal confessor. Her beliefs led her to order order the conversion or exile of their Muslim and Jewish subjects in Spain, in what would later become known as the Spanish Inquisition.
By 1491, members of both the Assassin and Templar Orders had infiltrated Ferdinand and Isabella's close circles. That year, the Genoese explorer Christopher Columbus, a close friend of Isabella's Jewish finance minister and secret Assassin Luis de Santángel, requested Isabella to fund his voyages to the East Indies. The Templars, aware that Columbus route would lead him to discover the New World, deliberately influenced Isabella into prolonging the ongoing war with the Moors, thereby preventing her from funding Columbus.
In 1492, the Italian Assassin Ezio Auditore da Firenze rescued King Muhammad XII from a Templar attack on his palace of Alhambra, and persuaded him into putting an end to the war with Ferdinand and Isabella. Queen Isabella, though grateful for Ezio's efforts, was still unable to finance Columbus' journey as her resources were still scarce. In addition to that, she revealed that the King of France had made Columbus an offer, though Ezio quickly realized it was a Templar trap and rescued Columbus. Luis de Santángel and Raphael Sanchez, Ferdinand's and Isabella's finance minister, eventually persuaded Isabella to fund half of Columbus' voyage, while they both paid the other half.
When Luis de Santángel died in 1498, the Assassins no longer had any eyes within the Spanish royal circle. In the early 1500s, Ezio Auditore sent a team of Assassins to retrieve Santángel's journal, resulting in the discovery that he had been trying to poison the Queen. Upon investigating whether Santángel's motives were out of revenge for the Spanish Inquisition which had killed most of his family or because of Templar influence, the Assassins found out that Isabella had been exchanging letters with Cesare Borgia, and concluded that she was forced into serving the Borgia. After establishing contact with Santángel's associate, one of Isabella's servants, the Assassins continued Santángel's work and started slowly poisoning the Queen.
In 1504, Ferdinand and Isabella made an arrangement with Pope Julius II to have Cesare Borgia imprisoned in the Castillo de la Mota near Valencia. However, Isabella later succumbed to the Assassins' poisoning on 26 November.