Reactivity-based one-pot synthesis of oligomannoses: Defining antigens recognized by 2G12, a broadly neutralizing anti-HIV-1 antibody

Article abstract of DOI:10.1002/anie.200353105

An HIV-1 vaccine may eventually be developed from oligomannoses synthesized by a reactivity-based one-pot self-condensation strategy. The compounds were tested for their ability to inhibit the binding of gp120 to the broadly neutralizing HIV-1 antibody 2G

Full text of DOI:10.1002/anie.200353105

Communications
Drug Development
Reactivity-Based One-Pot Synthesis of
Oligomannoses: Defining Antigens Recognized
by 2G12, a Broadly Neutralizing Anti-HIV-1
Antibody**
Hing-Ken Lee, Christopher N. Scanlan, Cheng-
Yuan Huang, Aileen Y. Chang, Daniel A. Calarese,
Raymond A. Dwek, Pauline M. Rudd,
Dennis R. Burton, Ian. A. Wilson, and Chi-Huey Wong*
The design of immunogens capable of eliciting broadly
neutralizing antibodies is a major, but elusive, goal of HIV
vaccine research.[1] However, a small panel of broadly
neutralizing human monoclonal antibodies (mAbs)[2] isolated
from seropositive donors may be very valuable in guiding the
design of such immunogens.[3] One antibody from this panel is
mAb 2G12, which recognizes a conserved and unusually
dense cluster of oligomannose residues on the “silent face” of
gp120, the envelope protein of HIV-1.[4]
The crystal structure of the Fab fragment of 2G12 shows
that the antibody adopts a highly unusual domain-exchanged
dimeric structure.[5] The structure produces an array of
antibody combining sites in proximity to one another
(Figure 1). Two conventional combining sites composed of
heavy (VH) and light (VL) variable domains sandwich an
unconventional site composed of residues from the two
neighboring VH domains.
The structure of Fab 2G12 complexed with Man9GlcNAc2
(1) shows that the conventional binding sites are occupied by
the D1 arms of the Man9GlcNAc2 moieties, and that the
terminal Mana1-2Man residues make 85% of the protein
[*] Dr. H.-K. Lee, C.-Y. Huang, Dr. A. Y. Chang, Prof. Dr. C.-H. Wong
Department of Chemistry and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2409
E-mail: wong@scripps.edu
C. N. Scanlan, Prof. Dr. D. R. Burton
Departments of Immunology and Molecular Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
D. A. Calarese, Prof. Dr. I. A. Wilson
Department of Molecular Biology and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Prof. Dr. R. A. Dwek, Dr. P. M. Rudd
Glycobiology Institute
Department of Biochemistry
University of Oxford
Oxford, OX1 3QU (UK)
[**] This work was supported by Optimer Pharmaceuticals, San Diego
(C.H.W.) and the National Institutes of Health (Grant no. AI33292
to D.R.B. and GM 46192 to I.A.W.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200353105
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thioglycosides are less reactive than other thioglycosides,
such as fucose and galactose.[6] In addition, 2-hydroxyman-
nose thioglycosides are in general much more reactive than
the corresponding 2-protected derivatives (Scheme 2), which
makes a one-pot synthesis with a universal leaving group
(used to simplify the protocol and conditions) difficult to
carry out. This problem was overcome by Ley and co-workers,
who synthesized trimannose with a one-pot strategy by
Figure 1. The structure of Man9GlcNAc2 (1; Man=mannose; Glc=glu-
cose; Ac, acetyl) on gp120 interacting with the Fab fragment of the
broadly neutralizing 2G12 antibody.
contacts. In the crystal, the D2 arms from two neighboring
Man9GlcNAc2 moieties occupy the nonconventional site.
We are attempting to use the structural information
obtained from the complexes of 2G12 with oligomannose
chains to design novel immunogens that will elicit a 2G12-like
antibody response. As part of these efforts toward develop-
ment of an HIV vaccine, we have designed and synthesized
the Mana1-2Man-containing oligomannoses 2–6 (Scheme 1)
by using a reactivity-based, modular one-pot synthesis
method that requires a minimal number of building blocks.
The programmable reactivity-based one-pot method for
oligosaccharide synthesis[6] has since been successfully
applied to the synthesis of several biologically significant
oligosaccharides, which include Globo H,[7] Lewis Y,[8] N-
acetyllactosamine oligomers,[9] and fucosyl-GM1.[10] Before
the one-pot synthesis could be applied to oligomannosides the
relative reactivity value (RRV) of each monomer had first to
be determined by a competitive HPLC assay. The RRV of
each mannoside is then used as a guide for the selection of
thioglycoside building blocks (Figure 2a).
An analysis of the RRVs of mannose building blocks for
the one-pot synthesis of trimannose Mana1-2Mana1-2Man,
the D1 arm of Man9GlcNAc2, suggests that d-mannose
Scheme 1. The structures of oligomannoses 2–6.
Figure 2. Strategy for a) sequential one-pot synthesis and b) one-pot self-condensation synthesis.
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Table 1: The reaction conditions used for reactivity-based one-pot self-
condensation of 7.
Entry T [8C] NIS [molar equiv][a] t [h] Yield of 8 [%] Yield of 9 [%]
1
À60 0.7
À50 0.7
À45 0.7
À40 0.7
À40 0.6
À40 0.5
À30 0.7
À20 0.7
24
24
2
1
1
0
0
5
2[b]
3
70
40
35
38
50
20
25
30
15
15
10
4
5
6
1
7
0.5 25
0.5
Scheme 2. RRV values of 2-hydroxymannose thioglycosides. Bn=ben-
8[c]
0
zyl, Tol=tolyl, TBDMS=tert-butyldimethylsilyl.
[a] NIS=N-iodosuccinimide. [b] The yield was calculated based on the
amount of 7 recovered. [c] Polymerization was observed.
changing the anomeric leaving group on mannose from F to
SePh to SEt, and thus changing the reactivity of this group.[11]
While we continue to find new protecting groups to tune
the reactivity of mannose thioglycosides for the one-pot
synthesis of oligomannoses, we report herein a new one-pot
strategy for the synthesis of both mannose dimer 8 and trimer
9. In our strategy, the most reactive monomer undergoes self-
condensation to give a less-reactive dimer. The dimer then
serves as an acceptor for another monomer molecule, which
leads to formation of the trimer (Figure 2b). Several 2-
hydroxymannose thioglycosides were designed and tested for
self-condensation but their reactions were not clean and
separation of the monomer, dimer, and trimer by column
chromatography was required. This problem was overcome
by introducing the nonpolar protecting group tert-butyldime-
thylsilyl to form 7, which undergoes self-condensation to give
dimer 8 and trimer 9 (Scheme 3).
Entry 5 in Table 1 lists the optimal conditions for the one-
pot self-condensation of 7 to produce 8 and 9. The reaction
temperature is key to the degree of self-condensation. No
self-condensation occurred at À608C (Entry 1) and mainly
dimer 8 and monomer 7 were found at À508C (Entry 2).
Uncontrollable self-condensation was observed at temper-
atures over À208C (Entry 8). The overall yield was reduced
when more than 0.6 molar equivalents NIS were added
(Entry 4), probably as a result of decomposition of the
thioglycosides, dimer 8, and trimer 9.
1111), which is then glycosylated by monomer 7 again to give
the least reactive molecule in the series, trimer 9 (RRV=
838). Subsequent removal of the TBDMS group and acety-
lation of 8 and 9 gave 10 and 11, respectively (Scheme 3).
Donors 10–12[12] were coupled to acceptor 13 or 14 (Bz,
benzoyl)[13] to give trimannose 15, tetramannose 16, penta-
mannose 17, heptamannose 18, or trimannose 19. Details of
these syntheses are shown in Table 2. Donors 10–12 exhibited
excellent Mana1-6Man or Mana1-3man selectivity as a result
of steric bulk at the 2-position of 10 and 11 and participation
of the acetyl group of 12. As shown in Table 2, deprotection of
oligomannoses 15–19 gave the corresponding deprotected
oligomannoses 2–6, respectively, in good yields.[9]
Man9GlcNAc2 (1)[5] and deprotected oligosaccharides 2–6
were evaluated for their ability to inhibit the interaction
between 2G12 and gp120 in an enzyme-linked immunosorb-
ent assay (see Figure 3).[4a] We will ultimately take the best
synthetic inhibitors found in this assay forward into experi-
ments to develop multivalent constructs as potential HIV
vaccine candidates for eliciting 2G12-like antibodies. The
results for trimannose 2 (15% inhibition at 2 mm) and
tetramannose 3 (79%) indicate that an extra a1-2 linked
mannose unit significantly enhances the inhibitory effect to a
level comparable with that achieved by Man9GlcNAc2 (71%).
This result is consistent with crystallographic studies that
identified the D1 arm of Man9GlcNAc2 as the primary
carbohydrate recognition motif for 2G12; compound 3
contains the same arrangement of mannose units as the
D1 arm. The Manb1-4GlcNAcb1-4GlcNAc core of
The RRVs of 7–9 were determined and, as expected,
monomer 7 (RRV= 8604) is the most reactive and undergoes
self-condensation to give the less-reactive dimer 8 (RRV=
Man9GlcNAc2 appears not to be critical for bind-
ing as it is absent from compound 3. Pentamannose
4 (79% inhibition at 2 mm), though lacking two
sequential Mana1-2Man units, is divalent which
may explain its more efficient inhibition of 2G12–
gp120 binding compared to that of compound 2
(15%). Surprisingly, heptamannose 5 (65% inhib-
ition at 2 mm), which contains both residues
mimicking the D1 arm of Man9GlcNAc2 and an
additional Mana1-2Mana1-2Mana1-6Man-linked
branch, does not offer a further increased affinity
over that of compound 3. The second branch, not
found in mammalian glycans, may force compound
5 to adopt an unusual conformation that is not
Scheme 3. One-pot self-condensation synthesis of building blocks 10 and 11. a) Tetrabutylam-
monium fluoride, tetrahydrofuran, RT, 24 h; b) Ac O, Et3N, 4-dimethylaminopyridine, CH2Cl2,
2
RT, 2 h.
optimal for 2G12 recognition.
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Keywords: antibodies · drug design ·
.
oligosaccharides · self-condensation ·
synthetic methods
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Table 2: The reaction conditions for the synthesis of oligosaccharides 15–18 and 2–6.
Donor Acceptor NIS [equiv] TfOH[a] [equiv] T [8C] t [h] Protected oligosac- Deprotected oligo-
charide (yield [%]) saccharide
(yield [%])
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10
11
10
11
12
13
13
14
14
14
1.3
1.3
2.6
2.6
2.6
0.13
0.13
0.26
0.26
0.26
À20
À10
À20
À10
0
2
4
2
4
15 (85)
16 (83)
17 (65)
18 (63)
2 (75)[b]
3 (72)[b]
4 (68)[c]
5 (65)[c]
6 (60)[c]
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24 19 (50)
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2
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Figure 3. Inhibition (%) of 2G12 binding to gp120. Man9=Man9Glc-
NAc2 (1).
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In conclusion, we have developed a novel and efficient
route to high-mannose oligosaccharides through a reactivity-
based one-pot self-condensation reaction. By using this
method we have prepared several Mana1-2Man-containing
oligosaccharides and found they effectively inhibit the bind-
ing of 2G12 to gp120. We have identified newsynthetic
epitope mimics that are as effective as, or better than,
Man9GlcNAc2 at inhibiting this interaction. Work is in
progress to develop multivalent constructs as candidates for
the development of an HIV vaccine.
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Received: October 16, 2003 [Z53105]
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