Aminoalkyl adenylate and aminoacyl sulfamate intermediate analogues differing greatly in affinity for their cognate Staphylococcus aureus aminoacyl tRNA synthetases

Article abstract of DOI:10.1016/S0960-894X(00)00360-7

Aminoalkyl adenylates and aminoacyl sulfamates derived from arginine, histidine and threonine, have been prepared and tested as inhibitors of their cognate Staphylococcus aureus aminoacyl tRNA synthetases. The arginyl derivatives were both potent nanomolar inhibitors of the Class I arginyl tRNA synthetase whereas for the Class II histidyl and threonyl tRNA synthetases, the acyl sulfamates were potent inhibitors but the adenylates had very little affinity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00360-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1871±1874
Aminoalkyl Adenylate and Aminoacyl Sulfamate Intermediate
Analogues Di€ering Greatly in Anity for their Cognate
Staphylococcus aureus Aminoacyl tRNA Synthetases
Andrew K. Forrest, Richard L. Jarvest,* Lucy M. Mensah, Peter J. O'Hanlon,
Andrew J. Pope and Robert J. Sheppard
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK
Received 7 April 2000; revised 16 June 2000; accepted 21 June 2000
AbstractÐAminoalkyl adenylates and aminoacyl sulfamates derived from arginine, histidine and threonine, have been prepared
and tested as inhibitors of their cognate Staphylococcus aureus aminoacyl tRNA synthetases. The arginyl derivatives were both
potent nanomolar inhibitors of the Class I arginyl tRNA synthetase whereas for the Class II histidyl and threonyl tRNA synthetases,
the acyl sulfamates were potent inhibitors but the adenylates had very little anity. # 2000 Elsevier Science Ltd. All rights reserved.
Aminoacyl tRNA synthetases are essential enzymes in
protein biosynthesis, catalysing the attachment of amino
acids to their cognate tRNA prior to delivery to the
ribosome. They are an ancient family of enzymes, lying
at the interface of the RNA and protein worlds, and have
been subject to considerable evolutionary divergence.
Consequently, selective inhibition of the prokaryotic
tRNA synthetases is attainable, as exempli®ed by the
clinical antibiotic mupirocin, and represents a strategy for
developing new classes of antibacterial agents. The
enzymes fall into two distinct classes which are char-
acterised by di€erent structural folds and sequence motifs.1
inhibitors of several tRNA synthetases and have found
many applications, from ligands for crystallography to
anity puri®cation.2 10 More recently, some aminoacyl
sulfamoyl adenosine (aminoacyl sulfamate) derivatives
(3) have been used in both mechanistic and crystal-
lographic studies.9 17 However, in many cases the inhi-
bitory potency of the aminoacyl sulfamates has not been
reported and there is very little comparative data for the
two inhibitor types. Here we report the synthesis of ami-
noalkyl adenylates and aminoacyl sulfamates derived
from some of the more highly functionalised amino acids,
arginine, histidine, and threonine, and their interaction
with their cognate aminoacyl tRNA synthetases.18
The aminoacyl tRNA synthetases catalyse a two stage
reaction in which the amino acid is activated as an ami-
noacyl adenylate intermediate (1), followed by attack of
the 20-OH or 30-OH of the tRNA on the activated carboxyl
group of the intermediate.
The acyl sulfamates were prepared by the general
method of Castro-Pichel et al.19 The protecting groups
chosen allowed facile unblocking of the functionality
using acidic treatments. Boc-Arg(Boc2)-OH was activated
as its imidazolide by reaction with carbonyl diimidazole
and coupled with the sulfamate (4)19 in the presence of
DBU (Scheme 1). The sulfamate (4) was treated simi-
larly with the N-hydroxysuccinimide esters of Boc-His
(Boc)-OH and Boc-Thr-OH to a€ord the protected
derivatives (5)±(7). Removal of the N-Boc moiety with
anhydrous TFA followed by addition of water to cleave
the O-isopropylidene group gave the required products
after puri®cation by HPLC.
The aminoacyl adenylate intermediate (Aa-AMP; 1) is a
high energy, hydrolytically labile, species. It is thus
tightly bound to the enzyme and stabilised analogues of
this intermediate have the potential to be potent inhibi-
tors. Aminoalkyl adenylates (2) have been described as
A phosphoramidite strategy was used for the prepara-
tion of (2a±c).10 The protecting groups were chosen to
allow deprotection using sequential basic and acidic
*Corresponding author. Tel.: +44-1279-627629; fax: +44-1279-622260;
e-mail: richard_l_jarvest@sbphrd.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00360-7

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A. K. Forrest et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1871±1874
treatments. The protected histidinol (12)20 was prepared
by activation of Boc-His(Boc)-OH with isobutyl chloro-
formate followed by reduction with sodium borohydride;
the other two protected amino alcohols were available
commercially (Scheme 2). Coupling of the protected
amino alcohols with the phosphoramidite (8)10 followed
by phosphorus oxidation gave the phosphotriesters (9)±
(11). Appropriate deprotection treatments furnished the
required aminoalkyl adenylates.
Table 1 along with reported values for the similar inhi-
bitors of S. aureus isoleucyl (IRS) and tyrosyl (YRS)
enzymes. Both the arginyl derivatives (2a) and (3a) were
potent inhibitors of arginyl tRNA synthetase (RRS) with
nanomolar potency. RRS is a Class I tRNA synthetase as
are IRS and YRS, and it can be seen that all three Class I
enzymes strongly interact with both aminoalkyl adenylate
and aminoacyl sulfamate intermediate analogues.
For the Class II enzymes HRS and TRS, the aminoacyl
sulfamate analogues are again potent inhibitors, although
the histidyl sulfamate is signi®cantly less potent against
HRS than the other sulfamate±synthetase pairs. How-
ever, in contrast to the Class I enzymes, the aminoalkyl
The analogues were evaluated in assays of the amino-
acylation activity of their respective Staphylococcus
aureus aminoacyl tRNA synthetase under conditions
normalised for substrate Km.21 The results are shown in
Scheme 1. Reagents: (i) Boc-Arg(Boc2)-imidazolide (from Boc-Arg(Boc2)-OH, carbonyl diimidazole, THF), DBU, THF, 1.5 h, rt; (ii) Boc-His
(Boc)-OSu, DBU, THF, 3 h, rt; (iii) Boc-Thr-OSu, DBU, THF, 24 h, rt; (iv) TFA, 0.5 h then TFA:H2O (5:1), 4 h, rt.

A. K. Forrest et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1871±1874
1873
Table 1. Inhibition of S. aureus cognate tRNA synthetases by inter-
mediate analogues
respectively. Although the more extended delocalisation
of the negative charge in the sulfamates may better com-
plement Class II transition state stabilisation, it seems
likely that the carbonyl group which is present in the acyl
sulfamate but not in the aminoalkyl adenylate, plays a
crucial role in recognition by the Class II enzymes.
Aminoalkyl adenylates (2)
Aminoacyl sulfamates (3)
Enzyme
No.
IC50 (nM) or
% inhibition
No.
IC50 (nM)
RRS
HRS
TRS
IRS
(2a)
(2b)
(2c)
Ð
7.5
20% at 300 mM
60% at 300 mM
780a
(3a)
(3b)
(3c)
Ð
4.5
130
15
Class I and Class II tRNA synthetases bind the aminoacyl
adenylate intermediate in di€erent conformations. In
Class I enzymes the carbonyl group is relatively exposed
Ð in Bacillus stearothermophilus tyrosyl tRNA syn-
thetase complexed with tyrosyl adenylate, the carbonyl
group does not appear to undergo any direct interaction
with the protein,6 as is also the case in the structures of
other Class I complexes with aminoacyl adenylate or sulfa-
mates. The aminoalkyl adenylates are thus able to act as
potent inhibitors of the Class I enzymes. By contrast, in
the Class II complexes, the carbonyl of the adenylate is
not only buried at the protein interface but it is also syn
to the adjacent amino group. There is a resulting net-
work of hydrogen bonds to the protein and to the
amine. For example, in the crystal structure of E. coli
HRS complexed to histidyl adenylate,22 the carbonyl
4a
YRS
Ð
11b
Ð
26b
aRef 9.
bRef 10.
adenylates have very little anity for either HRS or TRS.
The di€erence between the two inhibitor types is very
marked, with about four orders of magnitude di€erence
for the TRS inhibitors.
Both inhibitor classes exist in solution as the anionic form
of the acyl-phosphate mimic, due to the acidity of the
phosphodiester and of the acyl sulfamate NH function,
Scheme 2. Reagents: (i) Fmoc-Arg(PMC)-ol, tetrazole, 6.5 h then I2/H2O/2,6-lutidine, 2 h, rt; (ii) Boc-His(Boc)-ol, tetrazole, 5 h then I2/H2O/2,6-
lutidine 2 h, rt; (iii) Fmoc-Thr(tBu)-ol, tetrazole, 5 h then I2/H2O/2,6-lutidine, 2 h, rt; (iv) NH3, THF, H2O, 5±17 h, rt; (v) TFA 0.5 h then TFA:H2O
(5:1), 2 h, rt.

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A. K. Forrest et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1871±1874
group appears to participate in hydrogen bonds to
Arg113, Gln127 and to the alpha amino group. The dis-
ruption of this network of hydrogen bonds by replacing
the carbonyl with a methylene would account for the
large drop in anity of the aminoalkyl adenylate as
compared to the sulfamate.
10. Brown, P.; O'Hanlon, P. J.; Osbourne, N.; Mensah, L.;
Pope, A. J.; Richardson, C. M.; Walker, G. Bioorg. Med.
Chem. 1999, 7, 2473.
11. Ueda, H.; Shoku, Y.; Hayashi, N.; Mitsunaga, J.; In, Y.;
Doi, M.; Inoue, M.; Ishida, T. Biochim. Biophys. Acta 1991,
1080, 126.
12. Belrhali, H.; Yaremchuk, A.; Tukalo, M.; Larsen, K.;
Berthet-Colominas, C.; Leberman, R.; Beijer, B.; Sproat, B.;
Als-Nielsen, J.; Grubel, G.; Legrand, J.-F.; Lehmann, M.;
Cusack, S. Science 1994, 263, 1432.
13. Heacock, D.; Forsyth, C. J.; Shiba, K.; Musier-Forsyth,
K. Bioorg. Chem. 1996, 24, 273.
The relative anity of the aminoalkyl adenylate and
aminoacyl sulfamate inhibitors thus appears to re¯ect a
more general di€erence in the interactions with the high
energy intermediate of the catalytic cycle of the Class I
and Class II tRNA synthetases.
14. Cusack, S.; Yaremchuk, A.; Tukalo, M. EMBO J. 1996,
15, 6321.
15. Berthet-Colominas, C.; Seignovert, L.; Hartlein, M.;
Grotli, M.; Cusack, S.; Leberman, R. EMBO J. 1998, 17,
2947.
Acknowledgements
We thank Mr Martin Hibbs for enzyme puri®cation. This
work was partly supported by EU project PL962188.
16. Rath, V. L.; Silvian, L. F.; Beijer, B.; Sproat, B. S.; Steitz,
T. A. Structure 1998, 6, 439.
17. Bovee, M. L.; Yan, W.; Sproat, B. S.; Franklyn, C. S.
Biochemistry 1999, 38, 13725.
18. Sulfamate (3b) has been used as a tool compound but
neither its synthesis nor its inhibitory potency were reported.17
19. Castro-Pichel, J.; Garcõa-Lopez, M. T.; De las Heras, F.
G. Tetrahedron 1987, 43, 383.
20. Donner, B. G. Tetrahedron Lett. 1995, 36, 1223.
21. Compounds were assayed in a full aminoacylation assay
essentially as described previously for IRS and YRS.9,10 In all
cases the concentration of amino acid was equal to Km and the
ATP concentration was in excess (2.5 mM). For HRS23 and
TRS24 pure recombinant enzyme was utilised. For RRS par-
tially puri®ed native enzyme was used.
22. Arnez, J. G.; Harris, D. C.; Mitschler, A.; Rees, B.;
Francklyn, C. S.; Moras, D. EMBO J. 1995, 14, 4143.
23. Hodgson, J. E.; Lawlor, E. J. Eur. Pat. Appl., EP-785269,
(to SmithKline Beecham plc), 1997.
24. Hodgson, J. E.; Lawlor, E. J. Eur. Pat. Appl., EP-785271,
(to SmithKline Beecham plc), 1997.
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