Chemical synthesis of the hexasaccharide related to the repeating unit of the capsular polysaccharide from carbapenem resistant Klebsiella pneumoniae 2796 and 3264 †

The total synthesis of the hexasaccharide repeating unit of the CPS from carbapenem resistant K. pneumoniae 2796 and 3264 is reported using a sequential glycosylations approach. The total synthesis has been accomplished by glycosylation of rationally protected monosaccharide synthons derived from the commercially available sugars. The required uronic acid on the galactose moiety was successfully installed by a TEMPO-mediated late stage oxidation. The glycosylations were performed by the NIS-mediated activation of thioglycosides using H 2 SO 4 -silica as the promoter. Chloroacetate group was extensively used as a temporary protecting group to facilitate stereoselective glycosylations.


Introduction
Bacterial capsular polysaccharides (CPS) and lipopolysaccharides (LPS) are responsible for their virulence factor.CPS and LPS are made up of oligosaccharide repeating units with varied sugar residues.As they remain exposed on the outer surface of the bacterial cell wall, they play pivotal role in infection.Due to the presence of different sugar residues in the oligosaccharide repeats, CPS and LPS demonstrate diverse character and act as the elicitor of innate immune responses.2][3] However, it require tedious isolation and purication processes to harness these complex oligosaccharides in adequate quantity.Thus the chemical synthesis of the required oligosaccharides remains the only way to explore their vivid biological roles and potential as vaccine targets.
Klebsiella pneumoniae (K.pneumoniae) is an opportunistic pathogen that is responsible for community or hospital acquired infections.It mostly affects the urinary and respiratory tracts.Both CPS and LPS of the K. pneumonia are found to be responsible for the virulence.The CPS antigens are used for K-typing of K. pneumoniae whereas LPS antigens are used for O-typing. 4The capsular antigens are protective against capsular pathogens such as H. inuenza type b, meningococci and pneumococci. 5Among large number of K. pneumoniae Kantigens only a few are associated with human disease. 6This is a limiting factor for the development of suitable vaccine against this pathogen.Particularly the carbapenem resistant Kantigens (CRKP) are rarely known in the literature.Only recently, Kubler-Kielb has reported the structures of the CRKP CPS and LPS from clinical isolates collected from the infected patients of a CRKP outbreak in the US. 7Herein, we report the total synthesis of the hexasaccharide repeating unit of the CPS from K. pneumoniae 2796 and 3264 in the form of its p-methoxyphenyl glycoside (Fig. 1).The particular aglycon in the reducing end will enable us to form further glycoconjugates aer the selective removal of the same from the per-O-acetylated derivative of the target oligosaccharide.

Results and discussion
Judicious retro-synthetic analysis indicated that a sequential glycosylations strategy would be the best t for the successful synthesis of the target hexasaccharide 1.Thus, three rhamnose moieties were planned to be stitched by using the same synthon 5 with the reducing end galactose moiety 6.The chloroacetate group was thought to be the perfect choice as a temporary protecting group as it can ensure the required 1,2-trans glycosylations as well as can be de-protected selectively to pave the path for introduction of the next sugar unit.Next, a suitably protected galactose synthon 14 having a non-participating group at 2-position thought to be ideal for the required 1,2-cis linkage.A per-O-acylated rhamnose derivative 17 will complete the target hexasaccharide in its protected form.Finally, a TEMPO mediated oxidation of the primary hydroxyl group of the non-reducing end galactose moiety followed by global deprotection would furnish the required molecule (Fig. 2).
Finally, protection of the sole 2-OH with chloroacetate 11 group afforded the required donor 5 in 89% yield.Donor 5 was coupled with the known acceptor 6 (ref.12) by the activation of the thiotolyl using NIS in the presence of H 2 SO 4 -silica 13 at 0 C to afford the disaccharide 7 in 91% yield.It is worth noting that the use of H 2 SO 4 -silica as the promoter for NIS-mediated activation of the thioglycoside donor found to be benecial compared to the use of toxic, fuming and hygroscopic TfOH or TMSOTf.Further, selective de-protection of the chloroacetate group using thiourea 14 gave the disaccharide acceptor 8 in 87% isolated yield.Subsequently, glycosylations with the same donor 5 followed by de-protection of the chloroacetate group using the same reagent combination and condition was iterated twice to obtain the tetrasaccharide acceptor 12 (Scheme 1).The yields of the individual steps involved are mentioned in the Scheme 1.
In a separate experiment, known p-tolyl 2,3-di-O-benzyl-6-O-(4-methoxybenzyl)-1-thio-b-D-galactopyranoside (13) 15 was treated with chloroacetic anhydride in the presence of dry pyridine to give the completely protected donor ( 14) in 88% yield.Next, glycosylations of the donor 14 with the tetrasaccharide acceptor 12 using NIS in the presence of H 2 SO 4 -silica at À50 C gave the protected pentasaccharide 15 in 82% yield.Presence of the non-participating benzyl group at the 2-position of the galactosyl donor 14 and the reaction at very low temperature assured the formation of the desired 1,2-glycoside as the sole isolated product.Further, selective de-protection of the chloroacetate group using thiourea afforded the pentasaccharide acceptor 16 in 85% yield.Finally, glycosylations of 16 with the known donor 17 (ref.16) using the same NIS/H 2 SO 4silica at 0 C furnished the protected hexasaccharide 18 in 84% yield.At this stage, the strategically placed 4-methoxybenzyl group was selectively de-protected by oxidative cleavage using DDQ 17 to afford the hexasaccharide derivative 19 in 78% isolated yield.Oxidation of the primary hydroxyl group using TEMPO in the presence of bis-acetoxy iodobenzene (BAIB) 18 followed by catalytic hydrogenolysis and Zemplen de-O-acetylation 19 gave the target hexasaccharide 1 in 64% yield over three steps (Scheme 2).The amorphous white powder of compound 1 was triturated with CH 2 Cl 2 and ltered to remove aromatic impurities.

Conclusions
In conclusion, we have successfully accomplished the total synthesis of the hexasaccharide repeating unit of the CPS from K. pneumoniae 2796 and 3264 in the form of its p-methoxyphenyl glycoside.The practical synthetic strategy used the minimum protecting group manipulations and the chloroacetate group was used extensively as the temporary protecting group to ensure stereoselective glycosylations using rhamnose synthons.A TEMPO-mediated late stage oxidation was used successfully to generate the desired uronic acid moiety.The synthetic will denitely enhance the scope for further biological evaluation of the target oligosaccharide related to the carbopenem resistant K. pneumoniae strains and pave the path for a potential vaccine target.

Fig. 1
Fig. 1 Structure of the target hexasaccharide in the form of its pmethoxyphenyl glycoside.

Fig. 2
Fig.2Retrosynthetic analysis for the total synthesis of the target hexasaccharide 1.Scheme 1 Synthesis of the tetrasaccharide acceptor 12.