Siglec-2

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Discovery of Multifold Modified Sialosides as Human CD22/Siglec‑2 Ligands with Nanomolar Activity on B‑Cells Horst Prescher,† Astrid Schweizer,‡ Elena Kuhfeldt,† Lars Nitschke,‡ and Reinhard Brossmer*,§ †

G3-BioTec, 69207 Sandhausen, Germany Chair of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen,Germany § Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany ‡

S Supporting Information *

ABSTRACT: Sialic acids are abundant in higher domains of life and lectins recognizing sialosaccharides are heavily involved in the regulation of the human immune system. Modified sialosides are useful tools to explore the functions of those lectins, especially members of the Siglec (sialic acid binding immunoglobulin like lectin) family. Here we report design, synthesis, and affinity evaluation of novel sialoside classes with combined modification at positions 2, 4, and 9 or 2, 3, 4, and 9 of the sialic acid scaffold as human CD22 (human Siglec-2) ligands. They display up to 7.5 × 105-fold increased affinity over αMe Neu5Ac (the minimal Siglec ligand). CD22 is a negative regulating coreceptor of the B-cell receptor (BCR). In vitro experiments with a human B-lymphocyte cell line showed functional blocking of CD22 upon B-cell receptor (BCR) stimulation in the presence of nanomolar concentrations of the novel ligands. The observed increased Ca2+ response corresponds to enhanced cell activation, providing an opportunity to therapeutically modulate B-lymphocyte responses, e.g., in immune deficiencies and infections.

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conjugates, and liposomal formulations binding to CD22 are in development for the treatment of diseases like non-Hodgkin lymphoma, breast and prostate cancer, allergies, autoimmune diseases, and antidrug antibodies.23,24 The sialic acid binding site of CD22 is involved in the regulation of the B-cell receptor signal strength and BCR internalization.7,25,26 Ligands attached to antigens attenuate lymphocyte activation,26−29 whereas unconjugated ligands have been shown to enhance signaling.7,10 CD22 has been proposed as a target to speed up immune responses early in infection,30 and sialic acid-based ligands are suggested as molecules to enhance antibody production.7 Therefore, we believe high affinity CD22 ligands to be promising novel drug candidates to treat immune deficiencies and infections, especially those caused by sialylated pathogens.

ialic acids are heavily involved in cell−cell communications due to the exposed occurrence at the outermost end of oligosaccharides at the interface of the cell surface and extracellular space.1 They are as well exploited by many pathogens to attach to and infect cells, and moreover, many pathogens decorate themselves with sialic acids to escape the host immune system.2 Sialic acids perform many biological functions through interactions with sialic acid recognizing lectins.3 Isolation or synthesis of sufficient amounts of natural high affinity ligands to study the functions of such lectins or for therapeutic interventions is a major challenge. The many functional groups of sialic acid provide the opportunity to introduce affinity enhancing modifications giving high affinity, monovalent and selective ligands. Previously, the N-acetyl neuraminic acid scaffold was already modified at position 2, 5, or 9 to obtain first generation ligands for members of the Siglec (sialic acid binding immunoglobulin like lectin) family, some with sufficient affinity to study the lectin function.4−7 Monovalent and polyvalent ligands with higher affinity and selectivity were obtained by second and third generation ligands with double modification at positions 9 and either 2, 4, or 5 and triple modification at positions 2, 5, and 9.8−15 Here we present a new class of third generation ligands with simultaneous modification at positions 2, 4, and 9 as well as the next generation sialic acid lectin ligands with combined modifications at positions 2, 3, 4, and 9. The novel compounds display high affinity to human CD22 and are functionally active at low nanomolar concentrations in a cellular assay. CD22 belongs to the Siglec family and is strongly expressed on B-cells.16−22 Currently, several antibodies, antibody-drug © 2014 American Chemical Society

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RESULTS AND DISCUSSION Previously, we modified the minimal Siglec ligand scaffold 1 (αMe Neu5Ac) at position 9 and obtained the first synthetic Siglec ligands including first generation CD22 ligand 2 (BPC Neu5Ac, Figure 1).7 We were interested whether an additional substitution of 2 at position 4 would turn out to be beneficial for affinity toward CD22. Therefore, the 4-hydroxy group of 2 was selectively acylated to obtain the first generation of double substituted ligands for hCD22 (Supplementary Scheme S1). The compounds were tested in an ELISA-based hapten inhibition assay for their ability to block binding of hCD22Received: December 31, 2013 Accepted: April 2, 2014 Published: May 7, 2014 1444

dx.doi.org/10.1021/cb400952v | ACS Chem. Biol. 2014, 9, 1444−1450

ACS Chemical Biology

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We synthesized the 4-O-benzyl derivative 6 to test if the ether can replace the ester (Supplementary Scheme S2). Surprisingly, the affinity dropped by a factor of 80 compared to the 4-O-benzoyl derivative 5 (Table 1). These rIP values are opposite to those published for 4-O-acylated and 4-O-alkylated Siglec-4 (MAG) ligands,32 showing clear differences within the Siglec family. Identifying the carbonyl oxygen of the residue at position 4 as an important structural element for affinity to hCD22, we replaced the ester by an amide. Therefore, we directly replaced the 9-hydroxyl of 733 by an azide with a modified Appel reaction (Scheme 1) 34,35 and reduced azide 8 with triphenylphosphine in aqueous methanol with subsequent formation of the ammonium salt 9 (Scheme 1). This allows maintenance of the ester and provides high synthetic flexibility as shown for novel substitutions at position 9, e.g., alkylsulfonamide-based Siglec-7 ligands.36,37 The obtained amine 9 was biphenylated and directly converted into protected 11 via acetolysis.38 Introduction of the azide at position 439 was followed by attachment of the methyl glycoside and radical reduction of the azide40,41 to obtain amine 14. Acylation with various residues and final saponification (Scheme 1) led to an array of desired ligands with an amide at position 4. Importantly, the amide derivatives have similarly increased affinities as the ester derivatives (Table 2, 15−22), and again, we found small aliphatic residues to be most potent. Meanwhile, a very recent study showed the first second generation ligands with modifications at position 4 and 9, but different from our results, increased affinities to CD22 were reported only for substituted aromatic residues.8 The synthetic method is limited to reactive primary alcohols such as methanol, which complicates the combination with larger glycosides. Therefore, another method to combine positions 2, 4, and 9 was needed. Glycosylation of the 4-Namido-2-β-chloro sialic acids has been shown to lead to βglycosides,41 but 4-N-alkylated derivatives were not investigated in terms of their α/β selectivity, and no information about affinity toward CD22 was available. Regardless of this, we

Figure 1. Minimal Siglec ligand scaffold 1 (αMe Neu5Ac) with numbered backbone carbon atoms and CD22 ligand 2 (BPCNeu5Ac).

Fc protein to sialylated plate-bound IgM,31 and relative inhibitory potencies (rIP) were calculated using 2 as reference. A strongly increased potency, corresponding to an increased affinity to hCD22-Fc was found for aliphatic and aromatic residues (Table 1). Table 1. BPC-Sialosides with an Ester or Ether at Position 4

Scheme 1a

a

Synthesis of 9-BPC 4-NH-R derivatives: (a) TPP, CBr4, LiN3, DMF, 80%; (b) (i) TPP, MeOH, 3% H2O; (ii) HAc 20%, 98%; (c) 4biphenylcarboxylic acid, HATU, DIPEA, DMF, 92%; (d) H2SO4, HAc, Ac2O, 85%; (e) t-butanol, TMSiN3, quant.; (f) NBS, MeOH, 31%; (g) dioxane, tri-n-butyl tin hydride, 76%; (h) DIPEA, DMF, carboxylic acid anhydride or carboxylic acid 4-nitrophenyl ester; (i) EtOH, H2O, NaOH, two steps, 63−85%. 1445



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methyl derivative 23 (Table 2 and Supplementary Scheme S7) was obtained. The finding that the 4-N-morpholino α-glycoside 23 could be synthesized by a standard glycosylation reaction opens an interesting possibility to obtain α-glycans of other 4N-alkylated sialic acids. Compared to 2, the affinity toward hCD22 was essentially the same, indicating acceptance of a dialkylated amine at position 4 (Table 2, 23), although amides were more desirable for combination with larger glycosidic residues because of their higher affinity. It is possible to synthesize α-glycosides from protected 2,3-βepoxy sialic acid derivatives, but the influence of an adjacent azide at position 4 on synthetic procedures has not been described yet.43 In addition, a 3-equatorial hydroxyl remains within the molecule upon reaction of the epoxide, making the molecule more resistant to acidic conditions and neuraminidases,44 but this position was not yet investigated for Siglec ligands. To explore this alternative, we reacted glycal 12 with NIS under aqueous conditions to obtain 24, formed the 2,3-βepoxide 25, and opened the latter under acidic conditions in methanol analogous to known methods (Scheme 2).43,45 The adjacent azide did not exert a major influence, although fast formation of side products was observed under nonacidic conditions. A panel of amides was obtained by reduction of azide 26, acylation of amine 27, and subsequent saponification (Scheme 2 and Table 3 29−34). Importantly, the 3-hydroxyl group did not abolish binding, although the affinity was slightly lower than for the 3-H compounds. This is an important finding, as the 3-position can be easily modified. To further explore the possibility of replacing the amide at position 4 we synthesized some triazole, urea, and sulfamide derivatives (Supplementary Schemes S9 and S10). Triazoles showed decreased rIP values (Table 3, 36−38), whereas ureas and sulfamides were found to increase binding. Interestingly, the negatively charged N-sulfate 41 displayed so far the highest affinity (Table 3, 39−43). As we wished to combine residues at positions 2, 4, and 9, we screened in parallel for affinity improving glycosidic residues. Aromatic substitutions displayed only slightly increased binding, although the 2,3-dichlorobenzyl was described previously to improve binding substantially (Supplementary Schemes S14−S17 and Table S1).9 The saturated and more flexible propyl showed higher affinity than the less flexible allyl or the

Table 2. BPC-Sialosides with an Unsubstituted, Acylated, or Alkylated Amine at Position 4

introduced a morpholino group at position 4 analogous to a known procedure.42 Subsequent conversion to the glycosyl chloride S14 with AcCl/HCl was followed by introduction of the aglycone and saponification. Fortunately, the desired α-OScheme 2. Synthesis of 9-BPC 4-NH-R Derivativesa

(a) NIS, CH3CN, H2O, 60 °C, 70%; (b) CH3CN, DBU, 79%; (c) MeOH, camphor sulfonic acid, 91%; (d) MeOH, HCl, Pd, 60%; (e) DIPEA, DMF, carboxylic acid anhydride or carboxylic acid 4-nitrophenyl ester; (f) EtOH, H2O, NaOH, two steps, 53−86%.

a

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smaller methyl (Supplementary Schemes S11−S13 and Table 4). We modified the allyl glycoside with various substitutions

Table 3. BPC-Sialosides with Different Modifications at Position 4 and an Equatorial Hydroxyl at Position 3

Table 4. BPC-Sialosides with an Alkyl at Position 2

via thiol-ene coupling (Supplementary Scheme S13). Surprisingly, 51, containing an aliphatic chain and a terminal negative charge, has more than 100-fold improved binding. Analogue 52 without the thioether has a similarly high affinity (Table 4). We selected the 5-carboxypentyl glycoside for combination with residues at positions 3, 4, and 9. Epoxide 25 was reacted with 6-hydroxy hexanoic acid ethyl ester giving the desired glycoside (Supplementary Scheme S18). The yield was somewhat low (26%) due to formation of some β-glycoside as reported previously for larger alcohols in an analogous reaction43 and formation of side products. The obtained product was further reduced to obtain the amine and then acylated or sulfonylated and saponified in analogy to the methyl glycoside (Supplementary Scheme S18). The affinity assay showed indeed that the three additional substitutions at positions 2, 3, and 4 were tolerated and acted synergistically with about 1000-fold improved affinities (Table 5). The synthesis via the epoxide is relatively laborious and hampered by low yields, e.g., 7% overall yield in 5 steps from 12 to 55. Additionally, the 3-hydroxy group does not improve binding and is therefore dispensable. Aiming at analogues without a substitution at position 3, glycal 15 was reacted with hydrogen chloride to yield the 4-azido 2-β-chloro derivative (Supplementary Scheme S19) in a slightly modified procedure 1447



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the terminal negative charge contributes to enhanced binding remains so far elusive. To explore if the high affinity found in the ELISA assay has a consequence on B-cell signaling, compounds 53 and 57 were tested in a Ca2+ release assay employing the human Blymphocyte cell line Daudi as the model system. It was shown previously that 2 enhanced Ca2+ signaling in Daudi cells as well as in freshly isolated human blood lymphocytes upon stimulation with anti-IgM.7 Such BCR-stimulation results in Ca2+ release and subsequent cell activation and proliferation. Inhibitory properties of CD22 diminish this activation. However, blocking the inhibitory function of CD22 by CD22 ligands releases BCR signaling from this inhibition. While compound 2 can lead to an increased Ca2+ signal at 160 μM7 or as a dimeric compound at 12 μM,10 compounds 53 and 57 increased Ca2+ signaling consistently at concentrations of 400 nM (Figure 2). It should be noted, however, that the Ca2+

Table 5. BPC-Sialosides with Combined Substitutions at Positions 9, 4, 3, and 2

as used for the known 9-O-Ac analogue.46,47 Subsequent glycosylation with 6-hydroxy hexanoic acid ethyl ester gave the corresponding glycoside (Supplementary Scheme S19). Reduction of the azide, reaction with propanoic anhydride or SO3pyridine, and subsequent deprotection gave 55 and 56 (Supplementary Scheme S19). As expected, the affinity remained high (Table 6). As we found that 4-O-acyl and 4Table 6. BPC-Sialosides with Combined Substitutions at Positions 9, 4, and 2

Figure 2. Intracellular Ca2+ release assay. The human Burkitt lymphoma cell line Daudi was preincubated with PBS or CD22 ligands at indicated final concentrations and stimulated with anti-IgM. One representative experiment is shown out of 3 experiments with similar results.

measurement is only semiquantitative. In addition, CD22 is highly cis-masked on Daudi cells by cell surface bound sialic acids,48 quite different to the protein used in the affinity assay, which is free of CD22 masking sialic acids due to the expression in CHO-Lec-1 cells. The functional calcium release assay proves that the high affinity discovered with the ELISA assay indeed leads to a potent BCR-signaling modulation. Summarizing, we synthesized for the first time sialic acid αglycosides with combined modifications at positions 9, 4, 3, and 2. Such compounds provide a template for the discovery of ligands suitable for the large family of sialic acid lectins, especially human proteins like Siglecs as well as those of pathogens like viruses and bacteria.3 The novel compounds have up to 750 000-fold higher affinities to hCD22 compared to the unmodified sialic acid lectin ligand scaffold 1 (αMe Neu5Ac) (Table 7). Nanomolar concentrations were sufficient to functionally block the inhibitory function of hCD22 in a BCR-stimulation assay

N-acyl derivatives did not essentially differ in affinity, we asked whether this would be the same for the corresponding sulfates. Interestingly, 4-O-sulfate 57 turned out to be the most potent ligand with more than 3000-fold increased affinity compared to 2 (Table 6). So far we did observe important contributions to CD22 binding of a variety of substitutions in position 2 and 4. We cannot determine the exact source of energy gain upon binding to the protein due to lack of detailed structural information on the protein in complex with such ligands, but we presume involvement of additional hydrogen bonds to the 4-amide or 4sulfonyl oxygens to be most likely. The observed affinity increase of the negatively charged aglycone is at least in part due to hydrophobic interactions of the aliphatic chain, but how 1448



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sialosides enhance binding to Siglec-2 (CD22): towards potent Siglec inhibitors for immunoglycotherapy. Angew. Chem., Int. Ed. 52, 3616− 3620. (9) Mesch, S., Lemme, K., Wittwer, M., Koliwer-Brandl, H., Schwardt, O., Kelm, S., and Ernst, B. (2012) From a library of MAG antagonists to nanomolar CD22 ligands. ChemMedChem 7, 134−143. (10) Schweizer, A., Wöhner, M., Prescher, H., Brossmer, R., and Nitschke, L. (2012) Targeting of CD22-positive B-cell lymphoma cells by synthetic divalent sialic acid analogues. Eur. J. Immunol. 42, 2792− 2802. (11) Collins, B. E., Blixt, O., Han, S. F., Duong, B., Li, H. Y., Nathan, J. K., Bovin, N., and Paulson, J. C. (2006) High-affinity ligand probes of CD22 overcome the threshold set by cis ligands for binding, endocytosis, and killing of B cells. J. Immunol. 177, 2994−3003. (12) Abdu-Allah, H. H., Tamanaka, T., Yu, J., Zhuoyuan, L., Sadagopan, M., Adachi, T., Tsubata, T., Kelm, S., Ishida, H., and Kiso, M. (2008) Design, synthesis, and structure−affinity relationships of novel series of sialosides as CD22-specific inhibitors. J. Med. Chem. 51, 6665−6681. (13) Chen, W. C., Completo, G. C., Sigal, D. S., Crocker, P. R., Saven, A., and Paulson, J. C. (2010) In vivo targeting of B-cell lymphoma with glycan ligands of CD22. Blood 115, 4778−4786. (14) Shelke, S. V., Cutting, B., Jiang, X., Koliwer-Brandl, H., Strasser, D. S., Schwardt, O., Kelm, S., and Ernst, B. (2010) A fragment-based in situ combinatorial approach to identify high-affinity ligands for unknown binding sites. Angew. Chem., Int. Ed. 49, 5721−5725. (15) Mesch, S., Moser, D., Strasser, D. S., Kelm, A., Cutting, B., Rossato, G., Vedani, A., Koliwer-Brandl, H., Wittwer, M., Rabbani, S., Schwardt, O., Kelm, S., and Ernst, B. (2010) Low molecular weight antagonists of the myelin-associated glycoprotein: synthesis, docking, and biological evaluation. J. Med. Chem. 53, 1597−1615. (16) Pillai, S., Netravali, I. A., Cariappa, A., and Mattoo, H. (2012) Siglecs and immune regulation. Annu. Rev. Immunol. 30, 357−392. (17) von Gunten, S., and Bochner, B. S. (2008) Basic and clinical immunology of Siglecs. Ann. N.Y. Acad. Sci. 1143, 61−82. (18) Crocker, P. R., Paulson, J. C., and Varki, A. (2007) Siglecs and their roles in the immune system. Nat. Rev. Immunol. 7, 255−266. (19) Varki, A., and Angata, T. (2006) Siglecs: the major subfamily of I-type lectins. Glycobiology 16, 1R−27R. (20) Jellusova, J., and Nitschke, L. (2011) Regulation of B cell functions by the sialic acid-binding receptors Siglec-G and CD22. Front. Immun. 2, 96. (21) Walker, J. A., and Smith, K. G. C. (2008) CD22: an inhibitory enigma. Immunology 123, 314−325. (22) Tedder, T. F., Poe, J. C., and Haas, K. M. (2005) CD22: A multifunctional receptor that regulates B lymphocyte survival and signal transduction. Adv. Immunol. 88, 1−50. (23) Sullivan-Chang, L., O’Donnell, R., and Tuscano, J. (2013) Targeting CD22 in B-cell malignancies: Current status and clinical outlook. BioDrugs 27, 293−304. (24) Macauley, M. S., Pfrengle, F., Rademacher, C., Nycholat, C. M., Gale, A. J., von Drygalski, A., and Paulson, J. C. (2013) Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis. J. Clin. Invest. 123, 3074−3083. (25) Müller, J., Obermeier, I., Wöhner, M., Brandl, C., Mrotzek, S., Angermuller, S., Maity, P. C., Reth, M., and Nitschke, L. (2013) CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling. Proc. Natl. Acad. Sci. U.S.A. 110, 12402−12407. (26) Courtney, A. H., Bennett, N. R., Zwick, D. B., Hudon, J., and Kiessling, L. L. (2013) Synthetic antigens reveal dynamics of BCR endocytosis during inhibitory signaling. ACS Chem. Biol. 9, 202−210. (27) Courtney, A. H., Puffer, E. B., Pontrello, J. K., Yang, Z. Q., and Kiessling, L. L. (2009) Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation. Proc. Natl. Acad. Sci. U.S.A. 106, 2500−2505. (28) Duong, B. H., Tian, H., Ota, T., Completo, G., Han, S. F., Vela, J. L., Ota, M., Kubitz, M., Bovin, N., Paulson, J. C., and Nemazee, D. (2010) Decoration of T-independent antigen with ligands for CD22

Table 7. Relative Inhibitory Potencies (rIP) of the Novel Ligands Compared to Scaffold 1 compound

IC50 (μM)

rIP

1 (αMe Neu5Ac) 2 (BPC Neu5Ac) 57

1475 ± 935 6.11 ± 2.26 0.0020 ± 0.0006

1 240 755 000

with the human B-lymphocyte cell line Daudi. The compounds will be very useful to further investigate the function of CD22 on B-cells as well as on other human cell types expressing CD22 like dendritic cells,49 basophils,50 neurons,51 and mast cells.52 Further on, such ligands are promising tools for modulating human B cell responses in therapeutic settings, e.g., in immunodeficient patients and in infections with sialylated pathogens.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary tables and schemes; experimental details; biological methods; general chemical methods; detailed synthetic procedures; and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(R.B.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. Koch for excellent help with the affinity ELISA assay and H. Rudy and T. Timmermann for MS and NMR measurements, respectively. We are indebted to G. Fricker and A. Jäschke, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, for generously providing access to special institute equipment. We gratefully acknowledge the financial support of DFG (SFB643).

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REFERENCES

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