Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase

Article abstract of DOI:10.1021/acschembio.7b00601

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.

Full text of DOI:10.1021/acschembio.7b00601

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Article
Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural
Characterization with Adenine Phosphoribosyltransferase
Lawrence D. Harris, Rajesh K. Harijan, Rodrigo G Ducati,
Gary B. Evans, Brett M. Hirsch, and Vern L. Schramm
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00601 • Publication Date (Web): 27 Nov 2017
Downloaded from http://pubs.acs.org on November 29, 2017
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Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with
Adenine Phosphoribosyltransferase
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‡
‡
†§
Lawrence D. Harris* , Rajesh K. Harijan , Rodrigo G. Ducati , Gary B. Evans , Brett M.
‡
‡
Hirsch , Vern L. Schramm*
†
The Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd, Lower Hutt,
5
010, New Zealand
‡
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461,
USA
§
The Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
Auckland, New Zealand
L.D.H and R.K.H. contributed equally to this work.
Running title: Iminoaltritol Analogues of PRPP
Key Words: transition state analogues, enzyme inhibitors, stereochemical specificity, structure
of transition state analogue complexes, ribocation mimics
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Abstract
Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid
and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocationꢀlike
transition states with a partial positive charge residing on the pentose ring. Core chemistry for
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synthesis of transition state analogues related to the 5ꢀphosphoꢀαꢀDꢀribosyl 1ꢀpyrophosphate
(PRPP) reactant of these enzymes could be developed by stereospecific placement of bisꢀ
phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and
the bisꢀphosphates anchor both the 1ꢀ and 5ꢀphosphate binding sites. We provide a facile
synthetic path to these molecules. Cyclicꢀnitrone redox methodology was applied to the stereoꢀ
controlled synthesis of three stereoisomers of a selectively monoꢀprotected diol relevant to the
synthesis of transitionꢀstate analogue inhibitors. These polyhydroxylated pyrrolidine natural
product analogues were bisꢀphosphorylated to generate analogues of the ribocationic form of 5ꢀ
phosphoribosyl 1ꢀphosphate. A safe, high yielding synthesis of the key intermediate represents a
new route to these transition state mimics. An enantiomeric pair of iminoaltritol bisꢀphosphates
(L
ꢀDIAB and
falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine
phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of ꢀ and
DꢀDIAB) were prepared and shown to display inhibition of Plasmodium
L
Dꢀ
DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers
by altered ring geometry and bisꢀphosphate catalytic site contacts.
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Introduction
Essential steps in the synthetic pathways of purine and pyrimidine nucleotides, histidine and
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tryptophan, NAD , purine and pyrimidine base salvage, tetrahydromethanopterin synthesis and
other secondary metabolites depend on the transfer of the ribose 5ꢀphosphate group from 5ꢀ
phosphoꢀαꢀDꢀribose 1ꢀpyrophosphate (PRPP) to an acceptor molecule (1). Several
phosphoribosyltransferases have been subjected to transition state analysis by kinetic isotope
effects and quantum chemical analysis to reveal ribocationꢀlike transition states (2ꢀ5). The few
transition state analogues described for phosphoribosyltransferases have been focused on the
ribosylated product of the reaction rather than the PRPP ribosyl donor (5ꢀ8). We sought to
provide ribocation transition state mimics of PRPP as potential inhibitor probes for this relatively
untargeted class of important biosynthetic enzymes. Transition state analogues are chemically
stable mimics of the transition states of their cognate enzymes. For the PRPP transferases the
challenge is to make chemically stable mimics of PRPP, which is an intrinsically chemically
unstable molecule. Iminoribitols are known transition state mimics for ribosyltransferases
(Figure 1). Direct iminoribitol mimics of PRPP face a problem of chemical instability with
substitution of the anomeric carbon of ribose with both imino and pyrophosphate groups.
Therefore, we approached the synthesis by insertion of a methylene bridge between the anomeric
carbon and phosphate group, to generate bisꢀphosphorylated derivatives of 2,5ꢀdideoxyaltritol
(Scheme 1). The synthesis of
Dꢀ and Lꢀ2,5ꢀdideoxyꢀ2,5ꢀiminoꢀaltritol 1,6ꢀbisphosphate (DꢀDIAB
and ꢀDIAB) are described together with inhibition profiles for several
L
phosphoribosyltransferases and the crystal structures of Saccharomyces cerevisiae adenine
phosphoribosyltransferase (ScAPRT) in complex with both diastereoisomers.
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O
H
O
N
PNP
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NH
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H
NH
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NH
HO
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NH2
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OH OH
OH OH
OPO32-
OPO 2
-
OH OH
3
inosine
transition state
ImmH 56 pM TS analogue
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N
H
HGXPRT
O
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NH
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H
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NH
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NH
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HO PO
HO PO
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N
HO PO
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O
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NH2
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HOP O6
OH OH
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OH OH
OH OH
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OP O
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6
inosine 5'-PO4
ImmH 5'-PO 1 nM TS analogue
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transition state
PRTases
R-Acceptor
R-Acceptor
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-1
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HO PO
HO PO
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HO PO
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NH2
OPO3-2
-
3
OP O
2
6
OH OH
PRPP
OH OH
transition state
OH OH
-3
OP O
2
6
potential TS analogue
Figure 1. Ribocationic transition states for human purine nucleoside phosphorylase (PNP), Plasmodium
falciparum hypoxanthineꢀguanineꢀxanthine phosphoribosyltransferase, their transition state analogues and the
rationale for synthesis of the bisꢀphosphate iminoaltritols for other phosphoribosyltransferases (PRTases).
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Scheme 1. Targets and Retrosynthetic analysis.
Fiveꢀmembered cyclic nitrones add versatile functionality in the synthesis of cyclic amines. For
example, the syntheses of pyrrolidine alkaloids, azaꢀsugars and other natural products rely on the
robustness of the transformations available to these nitrones (9ꢀ13). A general strategy for the
synthesis of nitrones that allows facile access to many diastereoisomers of the iminosugar motif
has been the focus of several studies (14ꢀ16).
We have reported several strategies to incorporate 5ꢀmembered Nꢀheterocycles and iminosugars
into smallꢀmolecule, rationally designed, transitionꢀstate analogue inhibitors of
ribosyltransferases implicated in cancer, infectious diseases, gout and gastric ulcers (17). Here
we expand the approach to synthesize bisꢀphosphate DꢀDIAB (Scheme 1), a putative transitionꢀ
state analogue inhibitor of phosphoribosyltransferases. Inhibitors designed to inhibit activity of
glycosidases have occasionally shown surprising levels of activity for their enantiomers (18ꢀ21).
Thus, we also developed an efficient synthesis of enantiomer LꢀDIAB. We previously reported
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the synthesis of alcohol 2 (Scheme 1) with the 2,5ꢀcisꢀdihydroxymethyl configuration, from
iminoribitol imine 5a by addition of a lithiated diꢀthiane, hydrolysis and reduction (22). This is a
long and difficult synthesis. A facile route to 2 is desirable. We considered the previously
synthesized nitrone 6a (23) as a superior electrophile but safety is a concern in the use of
selenium dioxide/acetone/hydrogen peroxide (24) in its synthesis – especially for large scale
reactions. A nitrone/hydroxylamine/amine redoxꢀstrategy (12,25,26) (Scheme 1) would use
iminoribitol derivative 7 as a starting point for a number of stereoisomers and eliminate the need
to reꢀsynthesize each stereoisomer from different sugars (14).
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With the exception of a few literature examples (27) the 2,5ꢀtransꢀhydroxymethylꢀconfiguration
(cf. target DꢀDIAB), resulting from stereoselective additions to cyclicꢀnitrones or imines of this
type, has been installed in one of two ways: (i) where the configuration of the 3ꢀposition induces
the favored 2,3ꢀtransꢀrelationship (12) or (ii) where the configuration of the 2ꢀposition has been
inverted following addition of a suitable nucleophile. We envisioned a strategy employing reꢀ
oxidation (12,13,28) of a vinylꢀadduct of hydroxylamine after addition of Grignard (29ꢀ31) to
nitrone 6a, with subsequent oxidation/reduction (redox) steps, which could afford the desired
2
,5ꢀtrans-configuration in compound 4 (Scheme 1). The intermediate α,βꢀunsaturated
ketonitrone 3 in this case would doubleꢀup as a useful 2,5ꢀtransꢀhydroxymethyl (2,3ꢀcis–
hydroxymethyl) synthon. An alternative synthon for this purpose has been utilized by Marradi
and colleagues (28). However, the harsh conditions required for deprotection of this protectedꢀ
hydroxymethyl equivalent limits the synthetic utility of the synthon in subsequent
transformations; hence, we felt the vinyl group would allow for a wider range of functional
group manipulations. We report, herein, expedient syntheses of two enantiomeric bisꢀphosphates
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D
ꢀDIAB and
L
ꢀDIAB and improved synthesis of alcohol 2 from a common intermediate:
iminoribitol derivative 7.
We also investigated the effects on enzyme catalysis and structural properties upon binding of
the bisꢀphosphate transitionꢀstate analogue inhibitors to phosphoribosyltransferases adenine
phosphoribosyltransferase (APRT; EC 2.4.2.7) and orotate phosphoribosyltransferase (OPRT;
EC 2.4.2.10). These are examples of key enzymes in purine salvage and pyrimidine metabolism
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found among many organisms. The phosphoribosyltransferases catalyze the Mg ꢀdependent
reversible transfer of the 5ꢀphosphoribosyl moiety from αꢀ ꢀ5ꢀphosphoribosyl 1ꢀpyrophosphate
PRPP) to adenine or orotate, resulting in the formation of adenosine monophosphate (AMP) or
D
(
orotidine monophosphate (OMP) and inorganic pyrophosphate (PP ) (32ꢀ34).
i
RESULTS AND DISCUSSION
Synthesis of nitrone 6a (23), was substantially improved by implementing the catalytic
methylrhenium trioxide (MTO) (35ꢀ37) oxidation of secondary amines to oxidize iminoribitol 7
(Scheme 2). Nitrones 6a (72%) and 6b (18%) were easily separable by a combination of
recrystallization and chromatography in an excellent combined yield. We have successfully
performed the MTO oxidation on >20 g scale.
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Scheme 2. Synthesis of nitrones via MTO and improved synthesis of alcohol 2. Reagents and conditions: (i)
MTO (0.2 mol %), H O (30%) in CH Cl , r.t. then MeOH, 15 mins, r.t. then dropwise addition of 7 in CH Cl , 0
2
2
2
2
2
2
°C, 0.5 h, 72% for 6a and 18% for 6b (ii) Vinyl magnesium bromide, THF, –70 °C to 0 °C; (iii) Zn, AcOH, r.t., 4ꢀ16
h; (iv) Boc O, MeOH, r.t., 1 h, 85% over 3 steps for 12a or CbzCl, K CO , PhMe, H O, r.t., 1ꢀ3 h, 84% over 3 steps
2
2
3
2
for 12b; (v) OsO (4 mol %), NMO, MeCN, H O, r.t., 6 h; (vi) (1) NaIO , H O, EtOH, r.t., then (2) NaBH , r.t., 5
4
2
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2
4
mins to 1 h, 93% over 3 steps for 2a and 87% over 3 steps for 2b.
Nucleophilic addition of vinylꢀGrignard to nitrone 6a was highly efficient, yielding
hydroxylamine 10 (Scheme 2) quantitatively and with complete stereoselectivity as expected on
the basis of previous results (29ꢀ30). Reduction of the crude hydroxylamine afforded the free
secondary amine 11 which could be protected with Boc (12a) or Cbz (12b) groups. Catalytic
osmium tetroxideꢀmediated dihydroxylation of alkenes 12a and 12b, diolꢀcleavage using NaIO4
and reduction by NaBH , yielded diols 2a and 2b in 80% and 73% yields, respectively, over six
4
steps from nitrone 6a. The practical ease and enhanced safety profile of this route coupled with
the high yields marks a vast improvement on the previously reported synthesis of alcohol 2a
from iminoribitol 7 (22).
Next we turned our attention to the synthesis of bisꢀphosphate DꢀDIAB (Scheme 3). Conversion
of hydroxylamine 10 to the vinylꢀnitrone 3 was achieved in moderate yield by catalytic MTO
oxidation (36%) (38), and was improved to 49% by utilizing stoichiometric TEMPO as the
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oxidant (13), while a standard MnO oxidation protocol proved sluggish. Vinylꢀnitrone 3 was a
2
benchꢀstable solid.
Vinylꢀnitrones similar to 3 have been synthesized by modified Stille coupling of thioimidates
(39) or by aldolꢀtype reactions of αꢀmethylꢀsubstituted nitrones (40). The α,βꢀunsaturated
ketonitrone is an interesting and relatively unexplored functional group (41), whose chemistry
appears to be limited to addition to the ketonitrone functionality (42), cycloadditions (40) and
unusual rearrangement reactions (41). Here, the osmium tetroxideꢀmediated dihydroxylation of
the vinylꢀnitrone 3 with subsequent cleavage by periodate and reduction by sodium borohydride
yielded alcohol 13 with the desired configuration, presumably via the αꢀformylꢀnitrone species
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(43). Thus, vinylꢀnitrones ꢀ in this constrained bicyclic system ꢀ can furnish the αꢀhydroxymethyl
amine after this redox sequence. The isopropylidene unit stabilizes such systems (39), and we
have found that applying this type of nitrone redox chemistry to less constrained 5ꢀmembered
nitrogen heterocycles can be capricious. When vinylꢀnitrone 3 was treated with sodium
borohydride, saturated ethyl compound 19 was obtained indicating conjugate addition of hydride
to the vinylꢀnitrone had occurred. Presumably rapid tautomerization of the resulting eneꢀ
hydroxylamine intermediate regenerates the nitrone, albeit in low yield.
Scheme 3. Synthesis of bis-phosphate
DꢀDIAB. Reagents and conditions: (i) MTO (0.2 mol %), H O (30%) in
2 2
CH Cl , r.t. then MeOH, 15 mins, r.t. then dropwise addition of 10 in CH Cl , 0 °C, 0.5 h, 36%; (ii) TEMPO,
2
2
2
2
CH Cl , r.t., 10 mins, 49%; (iii) OsO (4 mol %), NMO, MeCN, H O, r.t., 6 h; (iv) (1) NaIO , H O, EtOH, r.t., then
2
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(
2) NaBH , r.t., 5 mins to 1 h, 47% over 3 steps; (v) Zn, AcOH, r.t., 4ꢀ16 h, quant.; (vi) Boc O, MeOH, r.t., 1 h,
4
2
74%; (vii) TBAF, THF, r.t., 85%; (viii) (1) N,N-diisopropyldibenzylphosphoramidite, CH Cl , r.t., 3 h, Ar(g) then
2
2
(2) m-CPBA, –40 °C to 0 °C, 1 h, 91%; (ix) H , 10% Pd/C, MeOH, r.t., 16 h; (x) MeOH, conc. HCl, r.t., 20 mins,
2
76% over 3 steps; (xi) NaBH , MeOH, r.t., 30 mins, 27%.
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Zincꢀmediated reduction of hydroxylamine 13, N-Bocꢀprotection and silylꢀdeprotection
furnished diol 16 in 63% yield over three steps (Scheme 3). To prepare the bisꢀphosphate ester
we chose to employ standard phosphoramidite coupling. Initially, double installation of diꢀtertꢀ
butyl phosphate was attempted via diꢀtertꢀbutylꢀN,Nꢀdiisopropylphosphoramidite with in situ
oxidation by mꢀCPBA, for a putative singleꢀstep, final, global deprotection. However, the
product contained impurities, some of which were identified as the Hꢀphosphonates (44). These
Hꢀphosphonates may have arisen from facile βꢀelimination of the intermediate phosphite species,
driven by the steric strain of the crowded environment of the 1,2,3ꢀcisꢀsetup. The installation of
the tert-butyl phosphate by exactly this method is entirely possible in a lessꢀcrowded
environment (5). Thus, benzyl phosphate esters were installed instead, by the same
phosphoramidite methodology. With the βꢀelimination pathway no longer available to the
intermediate dibenzylꢀphosphite, the bisꢀphosphate ester 17 could be prepared by this method
with a yield of 91%. Hydrogenation of the bisꢀdibenzyl phosphate ester followed by global
hydrolysis afforded bisꢀphosphate DꢀDIAB.
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0
Scheme 4. Synthesis of bis-phosphate
L
-DIAB. Reagents and conditions: (i) NaBH , MeOH, r.t., 30 mins, 96%;
4
(
ii) MTO (0.2 mol %), H O (30%) in CH Cl , r.t. then MeOH, 15 mins, r.t., then dropwise addition of 20 in CH Cl ,
2 2 2 2 2 2
0
°C, 0.5 h, 76% (recovered 21% of 20); (iii) Vinyl magnesium bromide, THF, –70 °C to 0 °C, 82% (combined
yield; Isolated yields: 46% for 21a, 31% for 21b); (iv) Zn, AcOH, r.t., 4ꢀ16 h; (v) Boc O, MeOH, r.t., 1 h, 70% over
2
2
steps; (vi) OsO (4 mol %), NMO, MeCN, H O, r.t., 6 h; (vii) (1) NaIO , H O, EtOH, r.t., then (2) NaBH , r.t., 5
4 2 4 2 4
mins to 1 h, 72% over 3 steps; (viii) TBAF, THF, 16 h, 72%; (ix) (1) N,N-diisopropyldibenzylphosphoramidite,
CH Cl , r.t., 3h, Ar(g), then (2) m-CPBA, –40 °C to 0 °C, 1 h, 58%; (x) H , 10% Pd/C, MeOH, r.t., 16 h; (xi)
2
2
2
MeOH, conc. HCl, r.t., 20 mins.
In order to synthesize bisꢀphosphate
L
ꢀDIAB, nitrone 6b was reduced with NaBH and
4
hydroxylamine 20 was isolated cleanly from the reaction mixture in 96% yield (Scheme 4). Once
again, this reduction was completely stereoselective. The MTO oxidation of hydroxylamine 20
yielded a chromatographically inseparable 1:1 mixture of nitrones 8 and 6b in 76% yield, along
with 21% of recovered starting material. Vinyl magnesium bromide was added to the inseparable
mixture 8/6b to afford a separable mixture of hydroxylamine adducts 21a/b in 82% combined
yield. Hydroxylamine 21a was reduced with zinc and N-Bocꢀprotected to afford alkene 23, in
70% yield over two steps, which was then dihydroxylated, cleaved and reduced to give alcohol
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2
1
4 in 72% yield over three steps. Silylꢀgroup deprotection of alcohol 24 by TBAF gave diol ent-
2
0
6 ([α]D –51.6 (c = 1.55, CHCl )), whose NMR spectroscopic data were identical to its
3
2
0
enantiomer 16 ([α]D +48.2 (c = 1.25, CHCl )). Elaboration of diol ent-16 to bisꢀphosphate Lꢀ
3
0
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0
DIAB was identical to the sequence described for bisꢀphosphate DꢀDIAB (vide supra).
Enzyme inhibition assays
Recombinant Nꢀterminal 6ꢀHisꢀtagged Saccharomyces cerevisiae (ScAPRT) was purified to
homogeneity using a NiꢀNTA Hisꢀtag affinity column, yielding approximately 60 mg of active
enzyme from 20 g of cell culture (45). Enzyme activity was assessed by use of a recently
published direct and sensitive fluorimetric assay, where 2,6ꢀdiaminopurine replaced adenine, the
natural substrate (Figure 2) (46).
Figure 2. The reaction catalyzed by ScAPRT was monitored on by the direct and sensitive decrease in fluorescence
signal as 2,6ꢀdiaminopurine is converted to 2ꢀaminoꢀAMP in the absence or presence of DꢀDIAB or LꢀDIAB.
Fitting the inhibition data to the equation for competitive inhibition yielded K values of 8.7 ± 1.5
i
µM for DꢀDIAB and 14.9 ± 2.9 µM for LꢀDIAB (Table 1). These K values are lower than the
i
previously reported 20 µM K for PRPP (45). Thus, both stereoisomers are good inhibitors,
M
binding better than the normal PRPP substrate.
The bisꢀphosphates
DꢀDIAB and LꢀDIAB were also assayed for inhibition of Plasmodium
falciparum (Pf) and human (Homo sapiens, Hs) orotate phosphoribosyltranferases (OPRTs).
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Both bisꢀphosphates inhibit PfOPRT and display 3ꢀfold selectivity for
DꢀDIAB (Table 1).
HsOPRT showed no apparent inhibition at
1).
DꢀDIAB or LꢀDIAB concentrations of 200 ꢀM (Table
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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4
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4
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5
5
5
5
5
5
5
6
0
1
2
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4
5
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7
8
9
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2
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5
6
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8
9
0
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2
3
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7
8
9
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2
3
4
5
6
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8
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2
3
4
5
6
7
8
9
0
Table 1. Inhibition data for bisꢀphosphates DꢀDIAB and LꢀDIAB. Initial reaction rates were used to calculate the Ki
values for both compounds in triplicate.
Compound
K
i
(ScAPRT/ꢀM)
K
i
(PfOPRT/ꢀM)
K (HsOPRT/ꢀM)
i
D
-DIAB
8.7 ± 1.5
39 ± 4
> 200
> 200
L-DIAB
14.9 ± 2.9
103 ± 17
Transition state analogues for purine nucleoside phosphorylases (PNPs), 5'ꢀmethylthioadenosine
phosphorylase (MTAP) and 5'ꢀmethylthioadenosine nucleosidase (MTAN) have also been
synthesized in distinct enantiomeric forms to examine their differential interaction with target
enzymes. In the cases of human PNP, MTAP and E. coli MTAN, the relative affinities were
1
,200, 9,600 and 2,250 more tightly bound, respectively, for the correct (3R,4S) configuration
than for the incorrect (3S,4R) enantiomers of tightꢀbinding inhibitors (47). ꢀ and ꢀDIAB differ
D
L
from tightꢀbinding inhibitors by only a 2ꢀ to 3ꢀfold binding discrimination for PfOPRT and
ScAPRT. A distinguishing difference between these inhibitor classes is that the former inhibitors
occupy the complete array of catalytic site interactions related to the transition state, while the
and ꢀDIAB molecules reflect binding only to the PRPP domain.
Structure determination and overall structure
Dꢀ
L
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ScAPRT crystallized from (NH ) SO had only sulfate at the active sites. DꢀDIAB and LꢀDIAB
4
2
4
binding was obtained by ligand soaking (SI). The molecular replacement using PHASER
(McCoy et al, 2007) for structure determination resulted in a dimer model in the C2 space group
0
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0
(
48). Electron density for the Nꢀterminal His tag was not observed in either structure. Electron
6
density was also not observed for the loop II (residues Glu104 to Asp110) of chainꢀB in the
DIAB bound structure and the same loop of chainꢀA of the ꢀDIAB bound structure. All other
amino acid residues were readily fitted into the electron density map. The densities of ꢀDIAB
and ꢀDIAB are clearly defined in the structures. All resolved amino acid residues are in the
most favored or are in the allowed regions of the Ramachandran plot (Table S2).
Dꢀ
L
D
L
ScAPRT belongs to the typeꢀI PRTs and is a homodimer (Figure 3 and Figure S1). Each
monomer consists of 5 αꢀhelices and 9 βꢀsheets. The enzyme has two domains, domain one
serves as a hood domain (residues 1 to 35) and a core domain (residues 36 to 178). The hood
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9
0
Figure 3: Subunit structures of ScAPRT complexes with
D
ꢀDIAB and
LꢀDIAB. (A) Structural fold and
superposition of the ScAPRTꢀ
D
ꢀDIAB (brick red) and ScAPRTꢀLꢀDIAB complexes (light blue). Loop I is from
Asn19 to Gly24, loop II is from Lys105 to Gly108 and loop III is from Asp163 to Lys165. (B) and (C) are structural
superposition of ScAPRTꢀ ꢀDIAB (brick red) and ScAPRTꢀ ꢀDIAB (light blue), respectively with (B) unliganded
ScAPRT (cyan) and (C) ScAPRTꢀsulfate bound (pink). (D) Superimposition of ScAPRTꢀ ꢀDIAB (brick red) and
ScAPRTꢀ ꢀDIAB (light blue) with ScAPRTꢀsulfate bound (pink), showing no significant loop movement in these
structures.
D
L
D
L
domain of ScAPRT contains αꢀhelices α1 (3ꢀ13) and α2 (31ꢀ35) and βꢀsheets β1 (14ꢀ18) and β2
25ꢀ30). The core domain of the enzyme consists of 5 parallel βꢀsheets (β3, 61ꢀ67; β4, 83ꢀ90; β7,
123ꢀ132; β8, 150ꢀ161 and β9, 173ꢀ178) and three αꢀhelices (α3, 37ꢀ54; α4, 69ꢀ81; and α5, 136ꢀ
48) (Figure 3 and S1). The loop between two of the βꢀsheets (β5, 97ꢀ104; β6, 109ꢀ115) of the
(
1
core domain is the catalytic loop (45). In the crystal structure, the transitionꢀstate inhibitors (Dꢀ
DIAB and LꢀDIAB) are bound at the active sites of both subunits of the ScAPRT (Figures 4 and
5
). Crystal soaking experiments with 1 mM adenine together with DꢀDIAB and LꢀDIAB gave
adenine bound only to chainꢀA and chainꢀB subunits, respectively (Figure 3 and 5). In the
adenineꢀfree sites, the symmetryꢀrelated Tyr107 side chain occupies the adenine binding pocket.
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No magnesium ion density was seen at the active sites of any of the inhibitorꢀbound structures,
despite the presence of 5 mM MgCl in the ligand soak solutions.
2
Binding of
ScAPRT with
active site with low Bꢀfactors, similar to the surrounding protein residues. The O2P of
hydrogen bonded to waterꢀ144 (Wat144). O3 makes hydrogen bond interactions with Wat60 and
D-DIAB, L-DIAB and adenine
0
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0
D
ꢀDIAB has been solved to 1.78 Å resolution (Figure S1).
D
ꢀDIAB is bound in the
ꢀDIAB is
D
(OD1)Asp130. O4 of
DꢀDIAB makes hydrogen bond interactions with Wat29 and
(OD2)Asp129. The O4P has hydrogen bond interactions with Wat88 and (OG1)Thr134. The
oxygen of O5P is hydrogen bonded to Wat24. Hydrogen bond partners for O6P include
OG)Ser137 and Wat213. The nitrogen atom N1 of the iminoꢀsugar ring has hydrogen bond
interactions with Wat203 (Figure 5).
(
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0
1
2
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4
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8
9
0
1
2
3
4
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6
7
8
9
0
Figure 4: Stereoview omit maps of
Dꢀ and
LꢀDIAB at the active sites of ScAPRT (A, B) and atomic numbering of
the enantiomers in the crystal structures (C, D). (A) The omit (Fo−Fc) difference electron density map of the
DIAB structure from subunit A. ꢀDIAB is added to the map as a stick model. (B) The omit (Fo−Fc) difference
electron density map of the ꢀDIAB structure with ꢀDIAB added as a stick model. The (Fo−Fc) difference maps
were calculated after 15 cycles of omit refinement by REFMAC5, leaving out the active site ligand. The contour
levels are at 2.5σ for the ꢀDIAB (PDB code 5VJN) and the ꢀDIAB complexes (PDB code 5VJP). Atomic
numbering of the ꢀDIAB (C) and (D) ꢀDIAM (D) used in the crystal structure contacts.
Dꢀ
D
L
L
D
L
D
L
L
ꢀDIAB in complex with ScAPRT was solved to 1.98 Å resolution.
ScAPRT active site with contacts similar to ꢀDIAB, but in the reverse orientation (Figure S1).
The interactions of ꢀDIAB to the enzyme are similar to the ꢀDIAB bound structure, except
there are fewer hydrogen bond interactions in the ꢀDIABꢀbound structure. The oxygen of O1P
is hydrogen bonded to Wat14. The O2P has hydrogen bond interactions with Wat85 and
OG1)Thr134. O3P is in hydrogen bond distance to (OG)Ser137 and (N)Ser137. O3 of ꢀDIAB
LꢀDIAB is bound to the
D
L
D
L
(
L
is in hydrogen bond interactions with Wat77 and (OD2)Asp129. O4 is hydrogen bonded to
Wat41 and (OD1)Asp130. The nitrogen atom N1 of the iminoaltritol ring has hydrogen bond
interactions with Wat19 (Figure 5). In the LꢀDIAB structure O4P, O5P and O6P of LꢀDIAB do
not make favorable hydrogen bond interactions with ScAPRT (Figure 5).
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The distance between the reactive N9 of adenine (or C9 in 9ꢀdeazaadenine) and the carbons of Dꢀ
DIAB and
LꢀDIAB corresponding to the reactive anomeric carbon of PRPP are an index of the
inhibitor fit at the catalytic site. ScAPRT complexes with adenine and
D
ꢀDIAB or ꢀDIAB can
L
0
1
2
3
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9
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2
3
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5
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8
9
0
be compared with human (PDB ID: 1ZN7) and Giardia lamblia APRTase (PDB ID: 1L1R) in
complexes with adenine (or 9ꢀdeazaadenine) and PRPP. The adenine binding mode is similar in
all the APRTase structures. In human APRTase the distance between N9 of adenine and reactive
C1´ of PRPP is 3.7 Å. This distance is within experimental uncertainty in the structure of adenine
with
distance is 3.4 Å; closer, presumably due to the transition state character of 9ꢀdeazaadenine.
However, when adenine and ꢀDIAB are bound to ScAPRT, the ring pucker of the complex
adjusts to the unfavorable configuration, causing the carbon corresponding to the anomeric
carbon of bound ꢀDIAB structures to be 4.4 Å from the anomeric carbon position. The carbons
DꢀDIAB in ScAPRT (3.8 Å). With 9ꢀdeazaadenine and PRPP in G. lamblia APRT, the
L
L
corresponding to the anomeric carbon (C1) of PRPP correspond to C2 and C5 of
DIAB, respectively (Scheme 1; Figure 4).
DꢀDIAB and Lꢀ
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Figure 5: Stereoviews of the active site of ScAPRT complexed with
D
ꢀDIAB (subunit A) and
ꢀDIAB, respectively. Hydrogen bond
interactions are shown by dotted lines. The red spheres are water molecules interacting with ꢀDIAB and ꢀDIAB.
(C) Superimposition of ScAPRT catalytic sites with bound ꢀDIAB and ꢀDIAB. The displacement of the iminoꢀ
LꢀDIAB (subunit B).
Panel (A) and (B) are the active site residues interactions with DꢀDIAB and L
D
L
D
L
altritol rings is apparent. The superimposed sulfate from the ScAPRTꢀsulfate structure shows its position in the
active site corresponding to the βꢀ1ꢀphosphate of the PRPP pyrophosphate.
Crystal soaks with the DIAB isomers and adenine reveal the adenine binding pocket adjacent to
D
ꢀDIAB (only in chainꢀA) and LꢀDIAB (only in chainꢀB). With DꢀDIAB, the adenine ring is
sandwiched between the hydrophobic side chains of Phe27 and Ile131. The exocyclic N6 amino
group is interacting with the peptide bond carbonyl of Leu26 and Wat14 while the N7 interacts
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with Wat191. The N7ꢀWat191 interaction is proposed to activate the N9 of adenine for attack of
C1' on PRPP in catalysis. In the Giardia lamblia APRT, N7 protonation for N9 activation role is
proposed to be due to a catalytic site Glu100 (49). Similar interactions are also observed in the
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ScAPRT complex with LꢀDIAB (Figure 5). Adenine binds to only one of the two subunits,
leaving the adjacent adenineꢀfree binding pockets occupied by a symmetryꢀrelated Tyr107 side
chain, suggesting subunitꢀsequential catalytic site activity.
Structural basis of ScAPRT inhibition
Although
and ꢀDIAB bind in reverse orientations with respect to the ScAPRT active site. The
configuration of the 2´, 3´, 4´ and 5´ carbons differs for the inhibitors, causing the iminoaltritol
ring to bind differently (Figure 5). Kinetic inhibition assays gave K values of 8.7 ± 1.5 ꢀM and
DꢀDIAB and LꢀDIAB bind to give similar interactions at the active site of APRT, Lꢀ
D
i
1
4.9 ± 2.9 ꢀM, respectively for
Interactions of ꢀDIAB to the active site of ScAPRT are slightly more favorable than
The N1, O4P, O5P and O6P of ꢀDIAB are not in hydrogen bond interactions, a possible
ꢀDIAB.
D
ꢀDIAB and
L
ꢀDIAB, despite the different binding modes.
D
LꢀDIAB.
L
explanation for the weaker binding of
L
Structural comparisons
The crystal structure of native unliganded (PDB ID: 1G2Q) and sulfateꢀbound structures (PDB
ID: 1G2P) are known (45). When compared to the unliganded ScAPRT structure (1G2Q), Loop
II (105ꢀ108) of the inhibitor bound structures is moved 5.8 Å towards the active site. A mutation
5
in this loop (eg. E106L) decreased k by 10 , therefore motion of Loop II toward the active site
cat
is forming the catalytically competent structure (45). Loop II is disordered in the sulfateꢀbound
structure (1G2P) (Figure 3 and S2). The loop between β1 and β2 (Loop I) of the hood domain
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retracts 1.5 Å and its position is distinct from the catalytic site. The loop from residues 160ꢀ168
(
Loop III) has an open conformation with inhibitors bound (5VJN and 5VJP) and in the sulfateꢀ
bound structure (1G2P), whereas this loop has a closed conformation in the unliganded structure
1G2Q). Loop III is moved 7.8 Å away from the active site in ꢀDIAB, ꢀDIAB and sulfate
1
1
1
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(
D
L
bound structures (Figure 3 and S2). These structural changes indicate that loops II and III are
important in catalytic site organization.
3
. Conclusions
We have highlighted the synthetic utility and flexibility of nitroneꢀredox methodology with
iminosugars to form informative inhibitors for phosphoribosyltransferases. The redox
methodology is particularly effective in the rigid, bicyclic system with the isopropylidene
protecting group in place, which allows completely stereoselective hydride reductions of the
nitrone group and imparts additional stability (35). As such, the commonly utilized iminoribitol 7
ꢀ
with a defined initial configuration ꢀ has led, with careful choice of redox and nucleophilic
addition strategies, to three different stereoisomers (2a, 15 and 24) of a selectively monoꢀ
protected diol. Two of these diastereoisomers (15 and 24) were elaborated to bisꢀphosphates
DIAB and ꢀDAIB, which were shown to display weak inhibition of human OPRT, significant
binding to PfOPRT and substantial inhibition of ScAPRT. The crystal structure of ꢀDIAB and
ꢀDIAB bound ScAPRT structures shows that both inhibitors bind to the enzyme active site with
D
ꢀ
L
D
L
favorable enzymeꢀinhibitor interactions. We have also described significant improvements to the
previously published synthesis of alcohol 2a (22,23), an intermediate utilized to prepare
numerous transitionꢀstate analogue inhibitors (17). In addition, compound 3 - an unusual vinylꢀ
nitrone species ꢀ has demonstrated functional utility as a trans-hydroxymethyl synthon in the
stereoselective addition to nitrone 6a.
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METHODS
Recombinant protein expression and purification
Recombinant Nꢀterminal 6× Hisꢀtagged ScAPRT was expressed and purified as previously
described (45), to >95% purity with modifications (see SI).
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ScAPRT inhibition assays
Enzyme activity assays for ScAPRT were performed at 25 °C in 50 mM HEPES pH 7.5 in 250
µL total reaction volumes, and each individual initial rate was the average of triplicate
measurements. Reaction mixtures contained 2 mM MgCl , 250 µM 2,6ꢀdiaminopurine (2,6ꢀ
2
DAP, fluorescent and easily quantitated product) (46), 250 µM PRPP, 1 mM DTT, and were
initiated by addition of ScAPRT (100 nM). DAP consumption was monitored by a change in
fluorescence (Ex: 280 nm / Em: 345 nm) over the course of 1 h using a SpectraMax M5 plate
reader (Molecular Devices), at variable concentrations of either DꢀDIAB or
Inhibition data for bisꢀphosphates ꢀDIAB and ꢀDIAB with OPRTs contained 500 ꢀM
orotidine 5’ꢀmonophosphate, 50 mM TrisꢀHCl, pH 8.0, 5 mM MgCl , 1 mM pyrophosphate, and
LꢀDIAB (0ꢀ200 µM).
D
L
2
2
5 nM PfOPRT or 200 nM HsOPRT at 25 °C. Reaction rates were monitored at 266 nm. Initial
reaction rates (triplicate) were used to calculate the K values.
i
Crystallographic inhibitor binding and structure determination
ScAPRT was used for the crystallographic inhibitor binding studies with DꢀDIAB and LꢀDIAB.
ꢀ1
Protein (10 mg ml ) was incubated with 2 equiv of
DꢀDIAB and LꢀDIAB separately for two
hours on ice. The coꢀcrystallization of the incubated proteinꢀligand mixtures were then screened
by the sitting drop vapor diffusion crystallization method at 22 °C using the Microlytic
(MCSG1ꢀ4) crystallization conditions as described in the SI and Table S1. The xꢀray diffraction
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data collection, data processing, structure determination, refinement and structure analysis are
detailed in the SI.
PDB ID Codes: The coordinates and structure factors are deposited at the Protein Data Bank
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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0
with codes 5VJN and 5VJP.
Corresponding Author Information: *Corresponding authors: lawrence.harris@vuw.ac.nz and
vern.schramm@einstein.yu.edu
Orcid id.: L.D.H. 0000ꢀ0002ꢀ4214ꢀ1018; R.K.H. 0000ꢀ0003ꢀ1503ꢀ5057; G.B.E. 0000ꢀ0002ꢀ
6
973ꢀ2002; R. G. D. 000ꢀ0002ꢀ8783ꢀ8847; V.L.S. 0000ꢀ0002ꢀ8056ꢀ1929;
Author Contributions: L. D. H. and G. B. E. conceived the synthetic routes and conducted all
chemical synthesis and characterization. R. K. H. conducted the crystallography experiments. R.
G. D. and B. M. H. purified target proteins and assayed inhibitors. V. L. S. conceived the
inhibitor experiments and interpreted results. All authors contributed to writing the manuscript.
Acknowledgements:
This work was supported by research grants from the New Zealand government (C08X0701) and
the National Institutes of Health research grants GM41916 and AI127807. The Einstein
Crystallographic Core XꢀRay diffraction facility is supported by NIH Shared Instrumentation
Grant S10 OD020068, which we gratefully acknowledge. This research used resources of the
Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEꢀ
AC02ꢀ06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRLꢀ
CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly
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6
Company, which operates the facility. Y. Zhang and R. G. Silva are acknowledged for the
enzyme preparations.
Supporting Information (SI) Available: This material is available free of charge via the internet.
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Supporting Information:
Enzyme preparation, crystallization and structure determination
Table S1. Crystallization and crystal handling
S2
S5
Table S2. Crystallographic data collection and refinement statistics
Figure S1. Structural folds of ScAPRTs.
S5ꢀS6
S7
Figure S2. Superimposition of ScAPRT complexes.
Scheme S1 and Supplementary Results and Discussion
Experimental section for chemical syntheses
S8
S9
S10ꢀ30
S31ꢀ86
NMR spectra for synthetic compounds
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L
ꢀDMDP, ꢀhomoDMDP and their Cꢀ3 fluorinated derivatives:
L
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