Synthesis and Characterization of a Linker for Primary Amines used in the Solid Phase Organic Synthesis of Spermidine

Neste trabalho é descrita a preparação de uma resina modificada, empregada como linker para a preparação de espermidina funcionalizada, em bom rendimento, assim como sua caracterização por infravermelho (IV), ressonância magnética nuclear de C no estado sólido com polarização cruzada e rotação no ângulo mágico (RMN-CPMAS de C) e ressonância magnética nuclear de H de alta resolução com rotação no ângulo mágico (RMN-MAS de H de alta resolução). O linker foi regenerado após clivagem da espermidina e reutilizado sem perda de eficiência.


Introduction
Spermidine is a naturally occurring polyamine, which like other amines as spermine and putrescine, is a growth regulator, besides possessing other unknown biological functions. 1In the last years a great deal of attention has been given to naturally occurring polyamines, especially because of their involvement in the regulation of cellular functions such as cell proliferation and differentiation.3][4] The inhibition of enzymes involved in the metabolism of parasitic protozoa has been recognized as a promising strategy for the chemotherapy of tropical diseases. 5,6Trypanothione, structurally characterized as N1,N8-bis(glutathionyl)spermidine, is a polyamine derivative used by trypanosomatids as a defense against reactive oxygen species during their infective cycle.The metabolism of trypanothione is a potential target for drug development against trypanosomiases and leishmanioses. 7,8s part of a research program aiming at the synthesis of polyamine derivatives, we became interested in the preparation of selectively protected spermidines. 9There are however many synthetic problems associated with polyamine chemistry, such as the amine differentiation and the ability to handle these exceptionally polar compounds.
To overcome these obstacles we decided to explore polyamine spermidine synthesis on a solid support with the use of a linker derived from dimethyl-barbituric acid.
Solid phase organic synthesis, SPOS, present established advantages over organic reactions in solution, [10][11][12][13][14][15] notably the possibility of using large excess of reagents and a simplification of the isolation procedures with no need of purifications in each step are among the most relevant ones.
7][18][19] A previous paper 20 described the synthesis of an orthogonally protected spermidine, using the Merrifield resin. 21In the present paper a linker resin prepared from dimethyl-barbituric acid and the glutaric anhydride was successfully used in the solidphase synthesis of a selectively protected spermidine.

Results and Discussion
Dimethyl-barbituric acid was transformed into 1,3-dimethyl-5-glutaryl-barbituric acid 1 by reaction with glutaric anhydride in THF at 81% of yield. 22his intermediate was linked to the polymeric resin using standard solid phase conditions by use of the diisopropylcarbodiimide (DIC) and dimethylamine pyridine (DMAP) to produce the linker resin 2 (Scheme 1). 23The aminomethyl resin was prepared from Merrifield resin, purchased from Aldrich, as previously described, 24 and Fmoc loading assay 25 showed a loading of 0.64 mmol g -1 .
Most compounds in Scheme 1 can be characterized by standard liquid-state methods, while for functionalized resins, as 2, adapted analytical techniques must be used to follow the reaction in the solid phase.Typically infrared spectroscopy [26][27][28][29] and solid state NMR techniques 10,[30][31][32] are proper to the task.Figure 1 illustrates the solid-state 13 C cross polarization magic angle spinning (CP/MAS) spectra 33 of compounds 1 and 2. The signal corresponding to COOH (at 182.5 ppm) is not present in the spectra of resin 2. Conversely, the formation of the N-C=O bond caused the broadening of the signal at d 170.2D TOCSY (total correlation spectroscopy) HR-MAS NMR 33 also confirmed the amide linkage (Figure 2).The resin 2 was then used in the synthesis of spermidine as demonstrated in Scheme 2. In order to initiate the preparation the 2-nitrossulfonamide was anchored on the resin 2 by reaction in THF under reflux. 34The resulting resin 3 then reacted with 4-bromobutylphthalimide in acetonitrile in the presence of Cs 2 CO 3 as base, generating the resin 4. 35 Protected spermidine 5 36 was cleaved from resin 4 after treatment with hydrate hydrazine at room temperature.Spermidine 5 was previously prepared by our group in solution system utilizing Fukuyama's sulfonamide. 37esin 2 was then reconstituted (Scheme 3) by treatment of resin 4, after cleavage of spermidine, with isopropyl nitrite in presence of H 2 O/AcOH in THF at room temperature overnight (Scheme 3).

Conclusion
In summary this communication reports a resin linker for the synthesis of functionalized spermidine.The resin was successfully prepared and characterized using solid state spectroscopic methods, infrared and NMR techniques Vol.22, No. 1, 2011   in phase solid proving the efficiency of this method.This linker was regenerated after cleavage of spermidine and re-used without loss of efficiency.It can be used similarly to other primary amines.

Scheme 1 .
Scheme 1. Synthesis of the linker resin.

Figure 2 .
Figure 2. High resolution solid state NMR spectra of the new linker (A) 2D TOCSY; (B) 1 H HRMAS; (C) 1D slice of 2D TOCSY spectrum showing the connection between the resin and the linker moiety.