Elsevier

Carbohydrate Research

Volume 346, Issue 17, 13 December 2011, Pages 2650-2662
Carbohydrate Research

Synthesis of a Forssman antigen derivative for use in a conjugate vaccine

https://doi.org/10.1016/j.carres.2011.09.015Get rights and content

Abstract

The total chemical synthesis of a Forssman antigen analog is described. The pentasaccharide contains a functionalized tether which should facilitate future conjugation with immunogenic proteins. We found that the total synthesis can be efficiently achieved by following a convergent 2+3 strategy, and using N-Troc protected GalNAc thioglycoside as a donor.

Introduction

The differential expression of carbohydrates on cell surfaces is widely recognized to be an integral component of many biological pathways and recognition processes. One such example is the glycolipid Forssman antigen, αGalNAc(1→3)βGalNAc(1→3)αGal(1→4)βGal(1→4)βGal(1→4)βGlc1-ceramide (1), an example of a sphingolipid which is an important class of glycolipids universally expressed in higher organisms such as humans, but typically not in lower organisms such as bacteria and yeast (Fig. 1). The Forssman antigen is a pentasaccharide member of the globo-series of glycosphingolipids, and very closely resembles the Globo H antigen as they share an identical Gb4 tetrasaccharide core.

Initial studies dating back to 1911 demonstrated that expression of the Forssman antigen is species specific: the antigen is expressed in some mammals (e.g., sheep, dogs, horses, chickens, etc.) but not in others (rabbits, pigs, humans, etc.), referred to as Forssman positive species and Forssman negative species, respectively.1 Forssman negative species instead express the tetrasaccharide precursor (Gb4), which is a substrate for the final enzyme in the biosynthetic pathway, a β(1→3)GalNAc transferase referred to as Forssman synthetase; the human genome contains the gene for this synthetase but it is not actively expressed.2 Together with playing an important role on the surface of Forssman positive mammalian cells as well as viral coats, interestingly the Forssman antigen is also present in several forms of human cancers (e.g., gastric, colon, lung).3 For individuals with Forssman positive tumors, chemical and immunological detection has confirmed the absence of this antigen on their healthy tissues.2 The identity of this pentasaccharide as a tumor-specific antigen makes it an interesting candidate for cancer therapy. An analogous target pentasaccharide which incorporated a 6-aminohexyl linker at the reducing end (2) was designed as a synthetic target; such a linker would facilitate conjugation with an immunogenic protein or peptide that would be capable of generating the desired T-cell dependant immune response (Fig. 1).

Although a few syntheses of the antigen were already available in the literature,4, 5, 6 none of the established methods had integrated a linker molecule onto the oligosaccharide. In addition, we sought to improve some of the limitations of the other methods. A chemical method was chosen over an enzymatic approach in order to be less restricted by the high substrate specificity,7, 8, 9 increased cost of glycosyl donors, and the limited number of glycosyltransferases available from commercial sources, commonly associated with enzymatic syntheses. A key consideration in the chemical synthesis involves the stereocontrol of the α- and β-linkages at the GalNAc residues. The glycan fragment of the Forssman antigen was first published by Paulsen et al. in 1980, in which glycosyl bromides and a 2+3 synthetic approach were utilized.4 This was followed by Ogawa’s group in 1989 synthesizing the glycosphingolipid in its entirety, utilizing an azide/silyl ether combination as a ceramide synthon and a glycopentaosyl fluoride donor.5 An alternative chemical synthesis was published by Magnusson et al. in 1994, which utilized AgOTf-mediated glycosylations to afford the pertinent α-linkage at the non-reducing end.6

Section snippets

Results and discussion

In an attempt to develop a more efficient chemical synthesis, we envisioned using an exceptionally stable glycosyl donor (as compared to Paulsen’s and Magnusson’s glycosyl halides) and to decrease the number of multistep functional group transformations that were present throughout Ogawa’s synthesis. Two different strategies were explored, an iterative 2+1+1+1 approach and a convergent 2+3 approach (Fig. 2). The first strategy relied on a 2,3-oxazolidinone group on the sugar at the terminal

Conclusions

Since the Forssman antigen is a tumor-specific antigen closely integrated with cancer biology, it is desirable to have it readily accessible in order to incorporate it into carbohydrate conjugate-vaccines used to target different types of cancer. It could become immensely valuable if able to effectively raise an immune response against the Forssman pentasaccharide expressed on the surface of cancer cells. Although improvements need to be made to the deprotection sequence, we have successfully

General methods

Optical rotations were determined in a 5-cm cell at 25 ± 2 °C. [α]D25 values are given in units of 10−1 deg cm2 g−1. Analytical TLC was performed on Silica Gel 60-F254 (E. Merck, Darmstadt) with detection by quenching of fluorescence and/or by charring with 5% sulfuric acid in water or with a ceric ammonium molybdate dip. All commercial reagents were used as supplied unless otherwise stated. Column chromatography was performed on Silica Gel 60 (Silicycle, Ontario). Organic solutions from extractions

References (28)

  • S. Nunomura et al.

    Tetrahedron Lett.

    (1989)
  • U. Nilsson et al.

    Carbohydr. Res.

    (1994)
  • J.B. Zhang et al.

    Carbohydr. Res.

    (2002)
  • J. Shao et al.

    Biochem. Biophys. Res. Commun.

    (2002)
  • L.J. Huang et al.

    Carbohydr. Res.

    (2006)
  • L. Yang et al.

    Carbohydr. Res.

    (2010)
  • J.H. Feng et al.

    Carbohydr. Res.

    (2010)
  • N. Tanaka et al.

    Carbohydr. Res.

    (2008)
  • U. Ellervik et al.

    Tetrahedron Lett.

    (1997)
  • R.B. Yan et al.

    Tetrahedron Lett.

    (2005)
  • J. Forssman

    Biochem. Z.

    (1911)
  • S. Hakomori et al.

    PNAS

    (1977)
  • K. Ono et al.

    J. Histochem. Cytochem.

    (1994)
  • H. Paulsen et al.

    Angew. Chem., Int. Ed. Engl.

    (1980)
  • Cited by (7)

    View all citing articles on Scopus
    View full text