The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation

Article abstract of DOI:10.1039/c9ob01436e

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2′-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2′-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.

Full text of DOI:10.1039/c9ob01436e

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Biomol. Chem., 2019, DOI: 10.1039/C9OB01436E.
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Number 47
21 December 2017
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The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MDAOIIT: 1)0c.1e03l9l/C9OB01436E
activation
Chriselle D. Braganza,a Kensuke Shibata,b,c* Aisa Fujiwara,b Chihiro Motozono,b,c Ko-hei
Sonoda,b Sho Yamasaki,c,d,e,f Bridget L. Stocker,a* Mattie S. M. Timmer.a*
a. School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600,
Wellington, New Zealand
b. Department of Ocular Pathology and Imaging Science, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan
c. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan
d. Laboratory of Molecular Immunology, Immunology Frontier Research Center, Osaka
University, Suita 565-0871, Japan
e. Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University,
Fukuoka 812-8582, Japan
f. Division of Molecular Immunology, Medical Mycology Research Center, Chiba University,
Chiba 260-8673, Japan
Abstract
Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T
lymphocytes that appear to play an important role in many pathologies ranging from viral and
bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the
presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR),
however few studies have explored the effects of systematic changes to the ligand structure
on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects
of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-ᴅ-
ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-
RU). Tetramer staining of MAIT cells revealed that the absence of the 2′-hydroxy group on the
sugar backbone of lumazines improved MR1–MAIT TCR binding, which could be rationalised

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using computational docking studies. Although none of the lumazines activated MAIT cells,
DOI: 10.1039/C9OB01436E
all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues
exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues
revealed different interactions between the sugar backbone and MR1 and the MAIT TCR
compared to those observed for the lumazines and confirmed the importance of the 2′-
hydroxy group for ligand binding and activity. Taken together, this information will assist in
the development of future potent agonists and antagonists of MAIT cells.
Introduction
Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T lymphocytes that
comprise 1-10% of peripheral blood T lymphocytes and up to 50% of liver T cells in humans.1,2
These cells bear a T cell receptor (TCR) with a highly conserved α chain (Vα7.2-Jα33 in humans,
Vα19-Jα33 in mice),3,4 and are activated by antigens bound to the major histocompatibility
complex (MHC) class I-like molecule MR1 on antigen presenting cells (APCs) (Figure 1).
Despite their high prevalence in humans, much remains unknown about the exact function of
MAIT cells, though they are considered to have key roles in immunity, particularly as MR1 is
highly conserved in mammalian species.5,6 MAIT cells are absent in germ-free mice,
highlighting the requirement of microbiota for their expansion,3, 7 and have been found to
migrate to inflammatory sites in diseases such as tuberculosis,8,9 multiple sclerosis,10,11
HIV,12,13 and type 114 and type 2 diabetes.15 Once activated via ligand presentation to their
TCRs, MAIT cells develop an effector phenotype and secrete pro-inflammatory cytokines,
proliferate, and can kill intracellular bacteria16,17 and/or modulate the development of
autoimmune diseases.18

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DOI: 10.1039/C9OB01436E
Figure 1. Ligand presentation by MR1 to MAIT cells
The ability of MAIT cells to respond to a variety of microbes in an MR1-restricted manner19
has led to much recent interest in identifying MAIT cell ligands and in understanding how
ligand structure influences the ensuing immunological response. The first ligand for MR1 was
identified by Kjer-Nielsen et al. in 2012 and determined to be 6-formylpterin (6-FP, 1a, Figure
2), a degradation product of folic acid (vitamin B9).20 This ligand and subsequently others
(e.g., acetyl-6-formylpterin [Ac-6-FP], 1b) were found to be potent antagonists of MAIT cells,
for while such ligands do not interact with the MAIT cell TCR, they can form a Schiff base with
the Lys43 residue of MR1.16 The first series of MR1 agonists, also identified by Kjer-Nielsen et
al.,20 were the ribityl lumazines 6,7-dimethyl-8-D-ribityllumazine (RL-6,7-diMe, 2), 7-hydroxy-
6-methyl-8-D-ribityllumazine (RL-6-Me-7-OH, 3a) and 6-hydroxymethyl-8-D-ribityllumazine
(RL-6-CH2OH, 4), which are products of riboflavin (vitamin B2) metabolism.20 These ligands
bind MR1 through a variety of aromatic and non-covalent interactions analogous to those
seen with 6-FP (1a), with the key difference being no Schiff-base formation but rather an
interaction of the ribityl moiety with the MAIT TCR through hydrogen-bonding.20,21
Subsequently, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU, 5a) and related
derivatives were identified as highly potent MAIT cell ligands due to their ability to form a
Schiff base with Lys43 on MR1 and interact with the MAIT TCR,17 with recent studies being
undertaken to develop more stable analogues of these compounds.22,23 A variety of drugs and
drug-like molecules were also recently screened as ligands for MR1, with direct contact of the
ligand with the MAIT TCR being required for agonist activity.24

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DOI: 10.1039/C9OB01436E
O
O
N
N
N
N
O
NHR1
NH
HN
N
OH
HN
N
OH
H
O
N
OH
O
N
OH
N
OH OH
OH OH
O
OH
1
2
3a
)
RL-6,7-diMe ( )
RL-6-Me-7-OH (
1a
R = H, 6-FP (
)
1
1b
R = C(O)CH3, Ac-6-FP (
)
O
N
O
HN
NH
HN
NH
OH
OH
O
O
N
OH
N
OH
H
N
OH OH
OH OH
O
OH
4
5a
)
RL-6-CH2OH ( )
5-OP-RU (
Figure 2. Representative MR1 ligands
Given the ability of the ribityl lumazines 2 and 3 to act as MAIT cell agonists, we became
interested in exploring how the substitution pattern of the sugar-motif influences MAIT cell
activation. This information, coupled with the knowledge about those structural motifs
required for irreversible binding to MR1,16-20,22,24 would aid in the design and synthesis of
potent MAIT cell agonists. However, the current synthetic route to the sugar-substituted
lumazines suffers from low overall yields (< 10%), and involves the synthesis of ribitylamine
through D-ribose-oxime formation and high pressure catalytic hydrogenation, followed by
condensation of the amine with 5-chloro-uracil, nitrosylation at the uracil 6-position and
reduction to form the corresponding 6-amino-uracil. Condensation of the diamine with either
diacetyl or pyruvic acid then forms RL-6,7-diMe (2) or RL-6-Me-7-OH (3a), respectively.20 Thus,
we first sought to develop an efficient three-step protecting-group-free synthesis of D-ribityl-
substituted lumazines RL-6,7-diMe (2) and RL-6-Me-7-OH (3a). Glyco-analogues of the
lumazine scaffold showing the greatest ability to bind MR1 would then be prepared using the
established route, which in turn would allow the effects of the sugar moiety on MAIT cell
activation to be explored. Given the recent interest in MAIT cells and the knowledge that their
activation, expansion, or suppression can play a key role in numerous diseases,11, 25-29 the
studies reported herein will therefore assist with the development of MAIT cell-mediated
therapeutics.

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DOI: 10.1039/C9OB01436E
Results and Discussion
The synthesis of RL-6,7-diMe (2) and RL-6-Me-7-OH (3a) began with the preparation of 1-
amino-1-deoxy-D-ribitol (7a) from D-ribose (Scheme 1) through a protecting-group-free
reductive amination reaction previously developed in our group.30 Here, D-ribose (6a) was
treated with NaCNBH3, NH4OAc (sat.) and NH3 (30% aq. soln.) in EtOH at room temperature
to smoothly afford the corresponding primary amine 7a in 82% yield following purification by
silica gel flash column chromatography. Nitration of 6-chlorouracil (8) with fuming HNO3 in
the presence of conc. H2SO4 afforded 6-chloro-5-nitrouracil (9) in 66% yield,31 which was
subsequently subjected to a substitution reaction with ribitylamine 7a under basic conditions
using aq. NaOH (pH 8) to give 5-nitro-6-ribitylaminouracil (10a) in a 74% yield.32 Next,
reduction of the nitro group in 10a using Fe powder in 10% AcOH (aq.), followed by
condensation with butane-2,3-dione under acidic conditions in a one-pot, two step procedure
afforded both RL-6,7-diMe (2) and its reduced form (11) in 65% combined yield. Here, it
should be noted that hydrolumazine 11 slowly reverted to the oxidised form when kept in
aqueous solution at room temperature. The same methodology was then applied to the
synthesis of RL-6-Me-7-OH (3a), whereby reduction of 5-nitro-6-ribitylaminouracil (10a) and
subsequent condensation with sodium pyruvate afforded the target compound in 40% yield
over the two-steps. Thus, we established a simple route to synthesise RL-6-Me-7-OH in 19%
overall yield, which was a significant improvement on the reported yield of < 10%.20

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DOI: 10.1039/C9OB01436E
OH
HO
NaCNBH3
NH3 (30% aq)
O
OH
OH
H2N
OH
OH OH
NH4OAc (sat)
EtOH, rt, 18 h
82%
HO
7a
6a
O
O
HNO3, H2SO4
66%
HN
NH
Cl
HN
NH
O
O
Cl
NO2
8
9
7a
, NaOH
EtOH:H2O (1:1)
74%
O
O
HN
NH
OH
HN
N
OH
Fe, 10% AcOH
O
N
OH
O
N
OH
O
H
NO2
OH OH
N
O
OH OH
(39%)
then
10a
2
O
Fe, 10% AcOH
O
+
then
ONa
HN
NH
OH
O
40%
OH
O
N
O
N
OH
N
OH OH
HN
11
(26%)
OH
O
N
OH OH
OH
N
3a
Scheme 1. Synthesis of lumazines 2, 3a and 11
Having demonstrated proof-of-concept and an efficient 3-step synthesis of the ribityl-
substituted lumazines, these ligands were then assessed for their MAIT cell activity.
Compounds were loaded onto empty MR1 tetramers33,34 and the resulting antigen-MR1
complexes tested for their ability to stain the 6C2 MAIT cell line5 and the TG40 cell line,35
which was used as a negative control as it lacks TCR-α and -β chains. The ligand 5-OP-RU (5a)
was used as a positive control for MR1-tetramer staining. Here, lumazines 2, 3a and 11 were
able to bind MR1 tetramers and stain the MAIT cells, as indicated by the co-staining of cells
bearing the T cell marker CD3ε (Figure 3). Of these, RL-6-Me-7-OH (3a) possessed the
strongest activity, with 96.4% of cells co-staining for both MR1-ligand complex and CD3ε. This
lumazine scaffold was then used to explore the effects of changes in the sugar moieties on
MAIT cell activation.

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DOI: 10.1039/C9OB01436E
Figure 3. MR1-ligand tetramer staining of MAIT cells. Empty APC-conjugated MR1 tetramers
were loaded with 5-OP-RU (5a) (positive control) and ligands 2, 3a, 11, and the ability of the
ligands to bind the tetramers determined using the MAIT cell line 6C2 and the parental cell
line TG40 (negative for TCR), and PE-conjugated anti-mouse CD3 for the identification of T
cells. Data is representative of three independent experiments.
With an efficient route for the synthesis of substituted lumazines in hand, the methodology
was then extended to derivatives of RL-6-Me-7-OH (3a) containing L-ribityl- (3b), 2-deoxy-D-
ribityl- (3c), D-arabinityl- (3d) and D-glucityl- (3e), rather than D-ribityl, sugar residues (Scheme
2). To this end, glycitylamines 7b-e were prepared in good yields (73-80%) via reductive
amination of the corresponding monosaccharides (6b-e), with subsequent substitution of the
chloride in 6-chloro-5-nitrouracil (9) with the different glycitylamines, using the
aforementioned methodology. Following in situ reduction of 10b-e and condensation with
sodium pyruvate, the target lumazines 3b-e were obtained in 18-27% yield over the three
steps.

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OH
OH
DOI: 10.1039/C9OB01436E
OH
(79%)
NaCNBH3
NH3 (30% aq)
, R =
, R =
, R =
b
c
d
e
OH
(80%)
OH OH
OH
OH OH
H2N
7b-e
R
R
O
NH4OAc (sat)
EtOH, rt, 18 h
OH OH
6b-e
OH
, R =
OH
(73%)
(77%)
OH
OH OH
O
O
O
, NaOH
7b-e
Fe, 10% AcOH
HN
N
HN
NH
Cl
HN
NH
O
O
N
R
then
O
O
N
R
EtOH:H2O (1:1)
H
ONa
N
NO2
NO2
OH
O
(76%)
(68%)
(72%)
(75%)
(35%)
(27%)
(37%)
(35%)
9
10b
10c
10d
10e
3b
3c
3d
3e
Scheme 2. Glyco-analogues of RL-6-Me-7-OH (3b-e)
The lumazines 3a-e were then assessed for their ability to bind MR1 and stain MAIT cells in
the MR1 tetramer assay. All sugar analogues of RL-6-Me-7-OH (3a) were able to bind MR1
and stain 6C2 MAIT cells to some extent (Figure 4), with the configuration of the 2′-OH in RL-
6-Me-7-OH (3a) proving unimportant for binding as the arabino-derivative 3d complexed to
MR1 was equably able to stain the cells. The 2-deoxy-D-ribityl analogue (dRL-6-Me-7-OH, 3c)
exhibited the highest level of staining, with 94.3% of cells co-staining for MR1-3c complex and
CD3ε. This observation was initially surprising as it had been previously demonstrated that
the ribityl 2′-OH of RL-6-Me-7-OH (3a) was involved in a hydrogen bond with the CDR3α Tyr95
residue in the crystal structure of an MR1–RL-6-Me-7-OH–MAIT TCR complex.21 MAIT cell
staining was largely abolished when using either L-ribityl analogue 3b or D-glucityl analogue
3e in the tetramer assay, indicating that the orientations of the 3′- and 4′-OH and the length
of the glycityl chain are important for MR1-MAIT TCR binding.

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DOI: 10.1039/C9OB01436E
Figure 4. MR1-glyco-analogue tetramer staining of MAIT cells. Empty APC-conjugated MR1
tetramers were loaded with 3a-e and assessed for their ability to stain the MAIT cell line 6C2
and the parental cell line TG40 (negative for TCR), and PE-conjugated anti-mouse CD3 for the
identification of T cells. Data is representative of three independent experiments.
To better evaluate the importance of the 2′-OH in the binding of lumazine ligands to MR1 and
the MAIT cell TCR, we docked dRL-6-Me-7-OH (3c) into the MR1-MAIT TCR complex. While
the crystal structure of mouse MR1-MAIT TCR is unavailable, the analogous human complex
is, and with high sequence identity with the mouse proteins (90% for the MR1-α1 and -α2
domains,36 and 72% for the MAIT TCR37), the hMR1-MAIT TCR complex serves as a suitable
substitute for our modelling studies. Moreover, it is also important to note that the CDR3α is
highly conserved in humans and mice, with identical contact residues between MR1 and the
MAIT TCR.21 The CDR3β loop, however, is more diverse.38
As can be seen from the docked structure (Figure 5), the lumazine of dRL-6-Me-7-OH is held
in the MR1 cleft by several hydrogen bonds and Van der Waals interactions, with overall
bonding being very similar to that previously noted for RL-6-Me-7-OH (3a).21
Notwithstanding, the 2′-deoxy-ribityl backbone and hydroxyls in 3c are oriented in a slightly
different conformation to that previously observed for 3a, thereby facilitating hydrogen
bonding to the Arg9, Arg94 and Tyr152 residues of MR1 (Figure 5A) as well as a hydrogen
bond between the 5′-OH and the CDR3α Tyr95 residue (Figure 5B). In the crystal structure of

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the RL-6-Me-7-OH (3a) complex, this hydrogen bond is present between the 2′-OH and
DOI: 10.1039/C9OB01436E
Tyr95.21 For dRL-6-Me-7-OH (3c), an additional hydrogen bond between the 5′-OH and CDR3β
Gly98 was also observed (Figure 5B). Although sequence variability in the CDR3β chain means
that not all TCRs will interact with MR1 in the same way, our observation that 3c forms a
hydrogen bond to the CDR3β backbone might indicate a measure of sequence independence
for MR1-ligand recognition by MAIT TCRs. Overall, it seems that the lumazine 3c can adopt an
alternative orientation within the MR1 cleft so as to optimise binding to the MAIT TCR, and
that the absence of a substituent at the 2-position may permit additional flexibility of the
sugar moiety, allowing for better binding interactions.
Figure 5. Docking of dRL-6-Me-7-OH (3c) into the hMR1-MAIT TCR complex (PDB: 4L4V). A.
Contacts between 3c and MR1. B. Contacts between 3c and MAIT TCR and MR1. Hydrogen
bonds are shown as black lines.
To assess the MAIT cell agonistic activity of lumazines 3a-e, the MAIT cell line 6C2 was
incubated with 3a-e and a mouse MR1-overexpressing NiH.cl9 cell line. 5-OP-RU (5a) and
antagonist Ac-6-FP (1b) were used as positive and negative controls, respectively (Figure 6A).
The expression of activation marker CD13739 was then assessed via flow cytometry. To our
surprise, at the highest tested concentration of 100 µM, none of the lumazines were capable
of activating MAIT cells. Instead, a significantly lower expression of CD137 was observed for
lumazines 3a-c, which indicated that these compounds may be acting as competitive
inhibitors similar to Ac-6-FP. To investigate the effect of 3c on 5-OP-RU activation, an

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inhibition assay was carried out whereby NiH.cl9 cells were incubated with Ac-6-FP (1b) or 3c
DOI: 10.1039/C9OB01436E
before stimulation with agonist 5-OP-RU and the addition of 6C2 cells (Figure 6B). A
concentration-dependent reduction in 5-OP-RU-mediated CD137 expression was observed
for both 3c and Ac-6-FP, however 3c was only capable of weakly inhibiting 5-OP-RU-mediated
MAIT cell activity. This may be due to the ability of Ac-6-FP (1b) and 5-OP-RU (5a) to form
Schiff bases with Lys43 of MR1, whereas lumazines 3a-e are only capable of forming non-
covalent bonds to MR1.21 Thus, while the 2-deoxy-D-ribityl-analogue 3c has the ability to
interact with MR1 and the MAIT cell TCR, this interaction is not sufficient to lead to MAIT cell
activation and instead leads to weak competitive inhibition of MR1. Hence, the generation of
a potent MAIT cell agonist would greatly benefit from the presence of electrophilic motif
capable of forming a covalent bond with MR1.21
Figure 6. MAIT cell activation and inhibition assays. A. The MAIT cell line 6C2 was cultured for
24 h on NiH.cl9 cells overexpressing mouse MR1 in the presence of 3a-e (100 µM) and the
mean fluorescence intensity (MFI) of CD137 expression was measured after gating on viable
TCRβ+ cells. 5-OP-RU (10 µM) and Ac-6-FP (100 µM) were used as positive and negative
controls, respectively. Data is representative of three independent experiments performed in
triplicate. (**) P ≤ 0.01; (***) P ≤ 0.001; (****) P ≤ 0.0001 compared to unstimulated cells. B.

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NiH.cl9 cells were incubated with 3c or Ac-6-FP (100 µM, 10 µM and 0 µM) for 1 h before the
DOI: 10.1039/C9OB01436E
addition of 5a (10 µM) and 6C2 cells for 24 h. MFI of CD137 expression was measured after
gating on viable TCRβ+ cells. Data is representative of two independent experiments
performed in triplicate.
Accordingly, we sought to prepare glyco-analogues of the potent covalent agonist 5-OP-RU
(5a) to investigate how changes to the sugar motif influence MAIT cell activation. Here, we
included additional derivatives containing the D-xylityl- (12f) and L-lyxityl- (12g) residues to
evaluate how systematic changes to the configuration of each hydroxyl group affects activity
(Scheme 3). We also included the D-allityl- (12h) residue as it contains the same configuration
as 5-OP-RU at the C2′-, C3′- and C4′-positions but contains an additional CH2OH group, which
may contribute to better activity via hydrogen bonding to residues in MR1 or the MAIT cell
TCR. As 5-OP-RU (5a) can be prepared in situ, through condensation of its precursor molecule
5-amino-6-D-ribitylaminouracil (5-A-RU, 12a) with methylglyoxal in the presence of MR1,17,
22,23 5-A-RU analogues 12a-h were prepared en route to the synthesis of the corresponding 5-
OP-RU analogues. To this end, the 5-nitro-6-glycitylaminouracils 10a-h were reduced under
the agency of sodium dithionite23 to afford the aminouracil HCl salts 12a-h in good yields of
58-71%.

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DOI: 10.1039/C9OB01436E
O
N
O
O
Na2S2O4, KOH
H2O
Methylglyoxal
HN
NH
HN
NH
HN
NH
In situ
O
N
R
O
N
R
O
N
R
H
H
H
NO2
NH2

HCl
O
10a-h
5a-h
12a
12b
12c
12d
12e
12f
12g
12h
(71%)
(67%)
(58%)
(63%)
(68%)
(63%)
(60%)
(61%)
OH
OH
OH
a
d
g
h
, R =
, R =
, R =
, R =
OH
OH
OH
OH
OH
OH OH
OH OH
OH
OH OH
OH OH
OH OH
OH
b
e
, R =
, R =
OH
OH OH
OH
OH OH
OH
OH OH
f
c
, R =
, R =
OH
OH
OH OH
Scheme 3. Glyco-analogues of 5-OP-RU.
For biological assessment, the 5-A-RU analogues 12a-h were converted to their 5-OP-RU
counterparts 5a-h via condensation with methylglyoxal immediately before testing against
the MAIT cell line 6C2. At a concentration of 10 µM, the D-arabinityl (5d), D-xylityl (5f), L-lyixityl
(5g) and D-allityl (5h) glyco-analogues exhibited significant levels of MAIT cell activity similar
to that caused by 5-OP-RU (5a) (Figure 7). Glyco-analogues 5b, 5c and 5e also caused
significant MAIT cell activation, albeit at lower levels than 5a. Here, the 2′-deoxy-D-ribityl
analogue 5c showed the lowest activity.

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DOI: 10.1039/C9OB01436E
Figure 7. MAIT cell activation assay for 5-OP-RU analogues 5b-e. The MAIT cell line 6C2 was
cultured for 24 h on NiH.cl9 cells overexpressing mouse MR1 in the presence of 5b-e (10 µM)
and the mean fluorescence intensity (MFI) of CD137 expression was measured after gating on
viable TCRβ+ cells. 5-OP-RU (10 µM) and Ac-6-FP (100 µM) were used as positive and negative
controls, respectively. Data is representative of three independent experiments performed in
triplicate. (***) P ≤ 0.001; (****) P ≤ 0.0001 compared to unstimulated cells.
To better understand these observations, we docked analogues 5b-h into the MR1-MAIT TCR
binding site and compared the binding interactions to those found in the crystal structure of
MR1-5-OP-RU complexed with a human MAIT TCR (Figure 8A). For all analogues, the planar
uracil ring adopted a very similar conformation to 5a when a covalent Schiff base was formed
with Lys43. However, the hydroxyl groups in each sugar analogue were oriented differently
and formed different sets of contacts with residues of MR1 and the MAIT TCR (Figure 8B). The
Tyr95 residue of the MAIT TCR showed interactions with the 2′-hydroxyl group of all the glyco-
analogues, with the exception of the 2-deoxy-D-ribityl (5c) and D-glucityl (5e) analogues.
Furthermore, the L-ribityl (5b) and 2-deoxy-D-ribityl (5c) analogues only made contacts with
Tyr95 at one position of the sugar, which could also contribute to the lower activity observed
with these analogues. These results indicate that for 5-OP-RU and its analogues, ligand activity

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depends on the number of interactions with Tyr95 of the MAIT TCR, and that an interaction
DOI: 10.1039/C9OB01436E
between the 2′-hydroxy group on the sugar backbone enhances MAIT cell activation. Taken
together, our results demonstrate that the MR1-MAIT TCR binding pocket can accommodate
changes to the sugar backbone, while still exhibiting ligand activity similar to 5-OP-RU.
However, none of the sugar derivatives showed greater activity than 5-OP-RU, suggesting that
the native D-ribityl chain in 5-OP-RU plays an important role in MR1-ligand-MAIT cell
recognition.
Figure 8. Docking of 5-OP-RU analogues 5b-h in the hMR1- MAIT TCR complex (PDB: 4PJ7). A.
Contacts between docked ligands containing sugar chains: L-ribityl (5b, light blue), 2-deoxy-
D-ribityl (5c, dark green), D-arabinityl (5d, light green), D-glucityl (5e, orange), D-xylityl (5f,
purple), L-lyxityl (5g, black) and D-allityl (5h, magenta) overlaid onto the crystal structure of 5-
OP-RU (5a, yellow) in the hMR1-TCR complex. Hydrogen bonds are shown as black lines. B.
Table listing the depicted hydrogen bonds.
Conclusion
We have developed a highly efficient 3-step synthesis of MR1 ligands based on the lumazine
scaffold. Key to this approach was the use of a protecting-group-free reductive amination
protocol, which also allowed for ready incorporation of modified sugar residues in the target
lumazines 3a-e and 5-OP-RU analogues 5a-h, and the first systematic analysis of the effect of
the sugar motif on MAIT cell activation. The results of this study indicate that although the
2-deoxy-D-ribityl-lumazine 3c can stain MAIT cells when complexed with MR1, 3c and the
other lumazines are unable to induce significant MAIT cell activation and instead act as weak

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competitive inhibitors of MR1. In contrast, different pentose and hexose analogues of 5-OP-
DOI: 10.1039/C9OB01436E
RU induced MAIT cell activation, albeit at lower or similar levels to 5-OP-RU (5a). Docking
studies revealed the importance of the 2′-hydroxy group on the sugar backbone for MAIT cell
activity, with the absence of the 2′-hydroxy enhancing MR1-MAIT TCR binding for the
lumazines, while this hydroxy group enhanced MAIT cell activation by the 5-OP-RU
derivatives. Taken together, these findings contribute towards an understanding of the
structural requirements for an MR1 ligand to activate MAIT cells and suggest that while
modifications to the sugar moiety can improve binding to MR1, a potent MAIT cell agonist
greatly benefits from the presence of the native D-ribityl chain along with an electrophilic
motif capable of forming a covalent bond with nucleophilic residues of MR1.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
BLS would like to thank the Health Research Council of New Zealand for financial support (HRC
Hercus Fellowship, 13/049). KS (2nd author) would like to thank JSPS for financial support
(Grant Number JP 16K08740) and Prof. Yasunobu Yoshikai (Kyushu University, Japan) for
providing the opportunity to undertake this work. KS (5th author) would like to thank JSPS for
financial support (Grant number 26293373). SY would like to thank MEXT (Grant number
26110009) and AMED (Grant number JP17gm0910010 and JP17ak0101070) for financial
support. We would like to thank Prof. Olivier Lantz for the provision of 6C2 αβTCR genes. CB
would like to thank Jordan McCone for help with the docking software and Makoto Seiki for
technical support.

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DOI: 10.1039/C9OB01436E
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