Asymmetric syntheses of L, L- and L, D-Di-myo-inositol-1,1′-phosphate and their behavior as stabilizers of enzyme activity at extreme temperatures

Full text of DOI:10.1002/anie.200900480

Communications
DOI: 10.1002/anie.200900480
Asymmetric Catalysis
Asymmetric Syntheses of l,l- and l,d-Di-myo-inositol-1,1’-phosphate
and their Behavior as Stabilizers of Enzyme Activity at Extreme
Temperatures**
Christina M. Longo, Yang Wei, Mary F. Roberts, and Scott J. Miller*
Di-myo-inositol-1,1’-phosphate (DIP) has been identified as a
major intracellular solute in several hyperthermophilic
organisms. In particular, a number of hyperthermophilic
microorganisms (including archaea and the bacterium Ther-
motoga maritima) accumulate DIP as an osmolyte when
grown above 758C;[1,2] the concentration of DIP increases
dramatically at temperatures greater than 808C.[3] The
stereoisomeric nature of naturally occurring DIP has been a
matter of discussion in the literature, with reports supporting
either the chiral l,l-DIP form, or its isomer, the meso l,d-
DIP form. Presently, most of the available data, including
biosynthetic[4] as well as biochemical and bioinformatic[5]
13C labeling and NMR spectroscopy studies.[4] One question
of interest, independent of the actual stereochemical assign-
ment for any material of natural origin isolated to date, is
whether or not the two stereoisomers are distinct functionally.
That is, do the two isomers exhibit differential abilities to
exhibit enzyme stabilization properties under extremophilic
conditions when examined with a unique enzyme derived
from a common organism? We now address this matter
directly through efficient, independent state-of-the-art chem-
ical syntheses of pure samples of known stereochemical
identity, along with preliminary biochemical studies with an
appropriate enzyme at high temperatures.
Previous research from our group has demonstrated that
histidine-based peptides can catalyze various enantioselective
phosphorylation reactions.[7] Shown in Scheme 1, the triol
2,4,6-tri-O-benzyl-myo-inositol (2) can be subjected to
desymmetrization using either of two enantiodivergent cata-
lysts (4 or 5) and diphenyl chlorophosphate to provide
phosphates 1 or 3 in good yields and excellent enantioselec-
tivity. We felt that catalysts 4 and 5 could thus be critical tools
for the independent synthesis of l,l-DIP and l,d-DIP, thus
enabling unambiguous access to these stereoisomers, and
therefore allowing direct comparison of their differential
prowess as stabilizers of enzyme activity under conditions of
high temperature.
studies, support the assignment of the l,d isomer of DIP as
a natural product. Importantly, it is also possible that different
organisms produce different stereoisomers as a part of their
individual metabolic capabilities. Through classical synthesis,
van Boom and co-workers suggested the l,l isomer could be
the naturally occurring form of DIP in Pyrococcus woesei,
based on comparison of the optical rotations of natural and
synthetic DIP.[6] On the other hand, in 2007 Santos and co-
workers suggested the natural material produced by Archae-
oglobus fulgidus is the l,d isomer of DIP based on
Our retrosynthetic plan was therefore quite straightfor-
ward (Scheme 2). In our approach, each inositol unit would be
subjected to phosphorylation in a sequential fashion, thus
ꢀ
allowing for rational control of O P bond formation at either
the 1-position (red) or the 3-position (blue). This plan
requires the synthesis of an optically pure, fully protected
inositol-derived phosphoryl chloride (6). Compound 6 could
then be coupled to 2, thus forming the desired phosphate
bond under the control of chiral catalysts such as 4 or 5.
Critically, phosphoryl chloride 6 contains a stereogenic
phosphorus atom. As a result, catalytic phosphorylation
reactions with peptide catalysts 4 and 5 create a fascinating,
but complex situation of double asymmetric induction, with
stereochemical preferences exerted by the catalyst and the
substrate.[8] Thus, we needed to establish if catalysts 4 and 5
could overcome any inherent substrate control in the
stereochemical outcome of the reactions (Scheme 3).
[*] C. M. Longo, Prof. S. J. Miller
Department of Chemistry, Yale University
225 Prospect Street, New Haven, CT 06520-4900 (USA)
Fax: (+1)203-496-4900
E-mail: scott.miller@yale.edu
Y. Wei, Prof. M. F. Roberts
Department of Chemistry, Boston College
Chestnut Hill, MA 02467 (USA)
Execution of the synthetic plan began with preparation of
phosphoryl chloride 6, and the recognition that it would not
likely be accessed in diastereomerically pure form. Further-
more, since myo-inositol phosphate esters with unprotected
hydroxy groups are prone to undesired cyclizations,[9] we
endeavored to alkylate the free hydroxy groups of 1
[**] We are grateful to the NIH (GM-068649 to S.J.M.) and to the
Department of Energy Physical Biosciences (DE-FG02-91ER20025
to M.F.R.) for support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200900480.
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Chemie
eters were evaluated (solvent, base, reagent ratios,
temperature, reaction time, and order of addition)
before settling on addition of phosphoryl chloride 6 to
triol 2 under the conditions shown in Scheme 4. These
studies were initially conducted with DMAP as an
achiral nucleophilic catalyst to define both optimized
conditions, and the inherent regioselectivity of the
process. The optimized conditions provided a mixture
of the two DIP adducts 8a and 9a (3:1) in a combined
yield of 57%.
Each isomer (8a and 9a) was separated and
carried forward to the corresponding isomer of DIP
in order to assign stereochemical identity. Notably,
reaction at the 1-position of 2 led to 8a, the precursor
to meso l,d-DIP, whereas reaction at the 3-position of
2 led to 9a, the precursor to optically active l,l-DIP.
As shown in Scheme 5, the remaining two hydroxy
groups of 8a and 9a were converted into benzyl ethers
to allow access to the meso or optically active
material. Optical rotation and 1H NMR data were
then used to determine that intermediate 8b was meso
(½aꢁ2D0 = 0, c = 0.4, D2O) and intermediate 9b was
optically active (½aꢁ2D0 = + 1.2, c = 0.4, D2O), thus
establishing the absolute stereochemistry of products
8a and 9a. Hydrolysis of the phenyl phosphate group
was then achieved using lithium hydroxide. Exchange
to the sodium salt and subsequent hydrogenolysis
yielded each isomer of the DIP product. The final
steps were exceptionally clean, enabling
Scheme 1. Desymmetrization of meso-triol 2. Boc=tert-butoxycarbonyl,
Bn=benzyl, Trt=trityl.
Scheme 2. Retrosynthetic analysis of DIP stereoisomers.
isolation of pure DIP with a minimum of
late-stage purification.
Having established the absolute config-
urations of products 8a and 9a, a prelimi-
nary study of chiral catalysts (e.g., 5 and 4)
used in the coupling reaction was under-
taken to assess if catalyst control might lead
to optimized yields of 8a and 9a, respec-
tively (Table 1). Achiral nucleophilic cata-
lysts such as DMAP and NMI (entries 1 and
2), defined the degree of substrate control
during the formation of the DIP precursors
(entries 1 and 2; l,l adduct/l,d adduct ꢂ 3–
Scheme 3. Projected peptide-catalyzed coupling reactions.
(Scheme 4). Reaction conditions that avoid for-
mation of cyclic phosphates were found to be
limited to neutral, acidic, or mild basic condi-
tions. In this vein, benzylation according to the
conditions of Poon and Dudley proved uniquely
successful.[10] Hydrolysis to the mono(acid) 7 was
then achieved with lithium hydroxide. Prepara-
tion of the inositol-based phosphoryl chloride
was then achieved using oxalyl chloride and
DMF. Within 5 minutes, chloride 6 was observed
as a mixture of diastereomers (2:1) that were
epimeric at the phosphorous center, as was
Scheme 4. Conditions and reagents: a) 2-benzyloxy-1-methyl-pyridinium triflate,
MgO, C6H5CF3; 73%; b) LiOH, THF/H2O (1:1); c) Dowex 50X2-200; quant. (over 2
steps); d) (COCl)2 (2 equiv), DMF (1 equiv), CH2Cl2, 5 min, RT; e) Et3N (4 equiv),
DMAP (1 equiv), 2 (2 equiv), RT, 12 h, 57%. DMAP=4-dimethylaminopyridine,
DMF=N,N-dimethylformamide, THF=tetrahydrofuran.
revealed in the 31P NMR spectrum.
We then turned our attention to the identifi-
cation of efficient reaction conditions for the
coupling of 2 and 7. Numerous reaction param-
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well for further study, and amplified instances of improved
peptides that could exhibit even higher degrees of catalyst
control in these reactions. It is further of note that these
differential ratios are achieved in a coupling reaction involv-
ing a diastereomeric mixture of phosphoryl chlorides, epi-
meric at the phosphorus atom, which necessarily contributes
to the complexity of these processes. To our knowledge,
preparation of diastereomerically pure phosphoryl chlorides
of this type is not yet reported in the literature.
With a robust synthesis of l,l-DIP and l,d-DIP secured,
we turned our attention to assessing the differential ability of
each isomer to contribute to thermoprotection in the context
of a key enzyme found in a unique extremophile. Archae-
oglobus fulgidus is one such organism. Therefore, its proteins
are good candidates to assess the effect of the solutes on
protein thermostability. Previously, it was shown that the
inositol monophosphatase (IMPase) from this organism
denatures irreversibly when heated above 908C.[4,5,12] This
enzyme thus serves as an interesting case to assess if the DIP
anion can reduce the extent of thermal denaturation.
Scheme 5. Conditions and reagents: a) 2-benzyloxy-1-methyl-pyridi-
nium triflate, MgO, C6H5CF3; 35%; b) LiOH, THF/H2O (1:1);
Chelex 100 Na form; 69% (over 2 steps); c) H2, Pd(OH)2/C, EtOAc/
MeOH (1:1); 96%; d) 2-benzyloxy-1-methyl-pyridinium triflate, MgO,
C6H5CF3; 27%; e) LiOH, THF/H2O (1:1; Chelex 100 Na form; 47%
(over 2 steps); f) H2, Pd(OH)2/C, EtOAc/MeOH (1:1); 82%.
For this study, we examined the effect of KCl and NaCl as
well as the glutamate salts of each cation (Figure 1) in
protecting the IMPase activity after heating to 958C for
15 minutes. This treatment in the absence of compatible
solutes rendered the enzyme nearly inactive (residual activity
was 3% after this heating). Thermoprotection could be
observed at 100 mm of added salt, although the effects were
much stronger at higher concentrations of the salts. Of
particular interest is the observation that the sodium salts
were virtually ineffective in this concentration range, while
KCl and K+/glutamate were similar and much more effective
at protecting IMPase activity.
4:1). The simple amino acid catalyst derived from histidine
(entry 3; with the p-nitrogen methylated, Pmh) revealed a
similar product ratio (3:1) as the achiral catalysts. However,
catalyst 5 (entry 4) showed a modest reversal (1:1.25) of the
inherent selectivity to favor the l,l-isomer (9a) slightly. On
the other hand, catalyst 4 (entry 5) exerted little stereochem-
ical influence and showed similar ratios in comparison to the
achiral catalysts. Investigation of 20 other catalysts[11] led to
the identification of a different catalyst (10) that gave
substantial selectivity for the l,d-isomer 8a (entry 6; 13:1),
and another (11) that more significantly reversed selectivity in
favor of the l,l-isomer 9a (entry 7; 1:2.5). These results bode
To get a measure of what each stereoisomer of the DIP
anion might contribute to thermoprotection, we examined the
effects of the Na+ salts of DIP (Figure 1, inset). We examined
Table 1: Examination of catalysts for selective synthesis of 8a and 9a.
Entry Catalyst sequence
l,d Adduct/
l,l adduct
1
2
3
4
5
6
7
4-dimethylaminopyridine (DMAP)
N-methylimidazole (NMI)
Boc-Pmh-OMe
5: Boc-Pmh-Asn(trt)-His(tBu)-Asp(OtBu)-Ala-OMe
4: Boc-Pmh-Hyp(tBu)-Sp5-Tyr(tBu)-Phe-OMe
10: Boc-d-Pmh-Pro-Aib-Trp(Boc)-Phe-OMe
11: Boc-Pmh-d-Pro-Aib-d-Phe-d-Phe-OMe
3:1
4:1
3:1
1:1.25
3.6:1
13:1
1:2.5
Figure 1. Effect of solutes on the residual activity of Archaeoglobus
fulgidus inositol monophosphatase, heated to 958C for 15 minutes,
+
&
then cooled and assayed at 858C: ~, KCl; ~, NaCl; , K /glutamate;
&, Na+/glutamate. The inset shows the effect of the sodium salts of
l,l- (*) and l,d- (*) DIP at 100 mm, as well as dimethylphosphate
+
^
(DMP; ꢀ) and myo-inositol ( ) compared to the K /salts solutes
(dashed lines) shown in the main figure.
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100 mm of each Na+/DIP on IMPase activity after heating. As
a control, myo-inositol at 100 mm was also examined for any
thermoprotection effects on this enzyme. Surprisingly, both
Na+/l,l-DIP and Na+/l,d-DIP were effective in producing a
recovery of activity (17% and 11%, respectively). In
comparison to the thermoprotection provided by KCl (18%
recovery of activity), it is particularly notable the DIP sodium
salts offer protection, when NaCl is completely ineffective,
therefore further pointing to the thermoprotective properties
of the DIP anions. Inositol alone had no effect. In addition,
the Na+ salt of dimethylphosphate (DMP) offered no
thermoprotective capacity, thus indicating that each DIP
anion on its own is unique and has significant thermoprotec-
tive effects.
The modest differences in the thermoprotective influence
of the two DIP stereoisomers are intriguing. At least in A.
fulgidus as well as in Thermotoga maritima,[13] the natural
product appears to be the l,d-DIP isomer. Yet, the fact that
the alternative stereoisomer also offers protective effects
raises fascinating questions about how the DIP anion
stabilizes proteins to thermal denaturation. While the molec-
ular details are not understood, the extended and rigid
network of the hydroxy groups on the inositol rings could help
to organize the water around the protein and stave off (to
some degree) irreversible unfolding.[14] The apparent lack of a
strong stereospecificity for these effects with the isomers of
DIP,[15] which have been elucidated by combined chemical
synthesis and biochemical studies, may provide an intriguing
and potentially unusual case of chirality in nature that is more
a matter of biosynthetic convenience rather than a matter of
evolutionary optimization. Our hope is that these chemical
and biochemical approaches will now set the stage for
detailed, high precision study of these intriguing phenomena.
[1] a) R. Cuilla, S. Burggraf, K. O. Stetter, M. F. Roberts, Appl.
Environ. Microbiol. 1994, 60, 3660 – 3664; b) L. O. Martins, H.
Santos, Appl. Environ. Microbiol. 1995, 61, 3299 – 3303.
[2] a) V. Mꢀller, R. Spanheimer, H. Santos, Curr. Opin. Microbiol.
2005, 8, 729 – 736; b) Y. K. Wang, A. Morgan, K. Stieglitz, B.
Stec, B. Thompson, S. J. Miller, M. F. Roberts, Biochemistry
2006, 45, 3307 – 3314.
[3] L. Chen, E. T. Spiliotis, M. F. Roberts, J. Bacteriol. 1998, 180,
3785 – 3792.
[4] M. V. Rodrigues, N. Borges, M. Henriques, P. Lamosa, R.
Ventura, C. Fernandes, N. Empadinhas, C. Maycock, M. S.
da Costa, H. Santos, J. Bacteriol. 2007, 189, 5405 – 5412.
[5] D. A. Rodionov, O. V. Kurnasov, B. Stec, Y. Wang, M. F. Roberts,
A. L. Osterman, Proc. Natl. Acad. Sci. USA 2007, 104, 4279 –
4284.
[6] S. H. van Leeuwen, G. A. van der Marel, R. Hensel, J. H.
van Boom, Recl. Trav. Chim. Pays-Bas 1994, 113, 335 – 336.
[7] a) B. R. Sculimbrene, S. J. Miller, J. Am. Chem. Soc. 2001, 123,
10125 – 10126; b) B. R. Sculimbrene, A. J. Morgan, S. J. Miller, J.
Am. Chem. Soc. 2002, 124, 11653 – 11656; c) B. R. Sculimbrene,
A. J. Morgan, S. J. Miller, Chem. Commun. 2003, 1781 – 1785.
[8] S. Masamune, W. Choy, J. S. Petersen, L. R. Sita, Angew. Chem.
1985, 97, 1 – 31; Angew. Chem. Int. Ed. Engl. 1985, 24, 1 – 30.
[9] D. C. Billington, The Inositol Phosphates: Chemical Synthesis
and Biological Significance, VCH, Weinheim, 1993.
[10] K. W. C. Poon, G. B. Dudley, J. Org. Chem. 2006, 71, 3923 – 3927.
[11] See the Supporting Information for details.
[12] This enzyme is involved in the biosynthesis of DIP, by removing
a phosphate monoester on one of the inositol rings of the DIP
precursor to generate the singly charged DIP. It will hydrolyze a
wide range of phosphomonoesters and is not specific as to the
rest of the molecule, see: K. A. Stieglitz, K. A. Johnson, H. Yang,
M. F. Roberts, B. A. Seaton, J. F. Head, B. Stec, J. Biol. Chem.
2002, 277, 22863 – 22874.
[13] H. Luk, M. F. Roberts, unpublished results.
[14] For example, two other hydroxylated phosphodiesters have been
detected in other hyperthermophiles: a-diglycerophosphate in
Archaeoglobus fulgidus under supraoptimal salinities and 1-
glyceryl-1-myo-inositylphosphate in Aquifex pyrophilius, see:
a) L. G. Gonꢁalves, R. Huber, M. S. da Costa, H. Santos, FEMS
Microbiol. Lett. 2003, 218, 239 – 244; b) P. Lamosa, L. G.
Gonꢁalves, M. V. Rodrigues, L. O. Martins, N. D. Raven, H.
Santos, Appl. Environ. Microbiol. 2006, 72, 6169 – 6173.
[15] S. Feng, S. L. Schreiber, J. Am. Chem. Soc. 1997, 119, 10873 –
10874.
Received: January 25, 2009
Revised: March 3, 2009
Published online: May 7, 2009
Keywords: asymmetric catalysis · extremophiles · inositols ·
.
peptides · phosphorylation
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