(+)-Zwittermicin a: assignment of its complete configuration by total synthesis of the enantiomer and implication of d-serine in its biosynthesis

Article abstract of DOI:10.1002/anie.200801561

(Chemical Equation Presented) D-Serine is the prime suspect: The total synthesis of (-)-zwittermicin A has been achieved from a suitably protected C₂-symmetric diamino tetraol unit and has allowed the complete stereochemical assignment ofnatural (+)-zwittermicin A. The unexpected 14R configuration implies the involvement ofa D-serine motifin the biosynthesis of (+)-zwittermicin A (see scheme).

Full text of DOI:10.1002/anie.200801561

Communications
DOI: 10.1002/anie.200801561
Natural Product Synthesis
(+)-Zwittermicin A: Assignment of its Complete Configuration by
Total Synthesis of the Enantiomer and Implication of d-Serine in its
Biosynthesis**
Evan W. Rogers, Doralyn S. Dalisay, and Tadeusz F. Molinski*
(+)-Zwittermicin A (1; Figure 1),[1] a water-soluble natural
antibiotic that was reported in 1994 and isolated from the
fermentation of the soil-borne bacterium Bacillus cereus,
carbon units (and subsequent loss ofCO 2), derived from
ꢀ
ꢀ
hydroxymalonate (HM; C7 C8), aminomalonate (AM; C9
ꢀ
C10), and the more common extender, malonate (C11
C12).[4]
Combinatorial biosynthetic engineering ofAM PKS
modules has great potential for the production of exotic
“non-natural” aminopolyketides[4c] and possible remodeling
ofPKS structures to alkaloids by exploiting the innate
nucleophilicity ofthe NH group. Despite high interest in 1,
2
the structure ofzwittermicin A has eluded attempts to define
its configuration for 14 years.[1c]
Herein, we report the complete absolute stereostructure
of( +)-1 by way ofdeductive reasoning and the ifrst total
synthesis ofits enantiomer ( ꢀ)-1. Our surprise finding—that
C13–C15 formally derives from d-Ser, rather than l-Ser[5]—
has implications for the structure–activity relationship of the
loading domain in the NRPS/PKS complex which initiates the
biosynthesis of( +)-1.
Figure 1. Natural (+)-zwittermicin A (1) and its enantiomer.
shows significant activity against phytopathogenic fungi.[2]
Most importantly, 1 synergizes the bioactivity ofthe endo-
toxin produced by Bacillus thuringensis (BT), a “green”
insecticide which is used globally for the protection of
Azidodiol 2, prepared from l-Ser as described earlier,[5]
was refunctionalized by protection of the terminal alcohol
with TBDPS,[6] protection ofthe secondary alcohol with
MOM,[7] and removal ofthe TBDPS group [6] to give 3 in high
yield (85% over 3 steps; Scheme 1).[8] Transformation of the
azido group of 3 into an N,N-dibenzyl group by hydro-
genolysis (Lindlarꢀs catalyst[9]) and subsequent benzylation at
the nitrogen center[6] gave a primary alcohol that was easily
oxidized to the stable aldehyde 4 (84% over 3 steps).
Evanꢀs aldol addition ofthe chiral glycolate equivalent
vegetable crops and for the eradication of gypsy moth from
[2,3]
forest trees.
BT toxin and related biocontrol agents are
important commodities used in the fight against declining
agricultural production and rising third world food short-
ages.[3b] The biosynthesis ofthe sugarlike natural product 1 is
very unusual; the molecule does not derive from carbohy-
drate metabolism, as the structure may suggest, but instead
arises from a non-ribosomal peptide synthetase/polyketide
synthase (NRPS/PKS) pathway that starts with an activated
serine unit (Ser; C13–C15, zwittermicin A numbering). Zwit-
termicin A is the first polyketide described in which the two-
carbon chain extensions occur by condensations ofthree-
5
[10] to 4 and subsequent removal ofthe chiral auxiliary under
standard reaction conditions afforded carboxylic acid 6 in
74% yield and 92% de (over 2 steps),[11] which is ready for
coupling to N-ureido-l-1,3-diaminopropionamide ((ꢀ)-8),
easily derived from the known amide 7.[12]
Coupling of 6 and (ꢀ)-8[13] gave the amide 9 (81%) which
was then globally deprotected[14] to afford (ꢀ)-10 with the
configuration proposed for (+)-1.[5] Although the 1H and
13C NMR spectra (400 MHz, D2O) of( +)-1[15] and (ꢀ)-10
were almost identical at C10–C15 (see the Supporting
Information), chemical shift differences at H8 [(ꢀ)-10, d =
4.53 ppm, d, J = 2.0 Hz; (+)-1, d = 4.56 ppm, d, J = 2.0 Hz]
were readily revealed upon analyzing the spectrum ofa
mixture ofthe two compounds (Figure 2 a). Additionally, the
specific rotation of (ꢀ)-10 ([a]D = ꢀ23.08, H2O) was opposite
in sign and oflarger magnitude than values measured ofr
natural (+)-1 ([a]D = + 8.18, H2O; lit.[1a] + 8.98, H2O) under
the same conditions.
[*] E. W. Rogers, D. S. Dalisay, Prof. Dr. T. F. Molinski
Department of Chemistry and Biochemistry
and Skaggs School of Pharmacy and Pharmaceutical Sciences
University of California, San Diego
9500 Gilman Drive, La Jolla, CA, 92093-0358 (USA)
Fax: (+1)858-822-0386
E-mail: tmolinski@ucsd.edu
[**] Financial support for this work was provided by the National
Institutes of Health (RO1 AI039987). We are grateful to Prof. T. M.
Zabriskie (Oregon State University) and Prof. M. Burkart (UC San
Diego) for helpful discussions. HRMS measurements were carried
out by R. New (UC Riverside) and Y. X. Su (UC San Diego). We thank
D. Manker (Novo Nordisk, Entotech Inc, Davis, CA) for a gift of
authentic (+)-1.
The relative configuration ofthe C8–C15 segment of( +)-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801561.
1 was certain from the analysis of 1H NMR spin system
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Figure 2. 1H NMR spectra (400 MHz, D2O): a) 1:3 mol ratio of syn-
thetic (ꢀ)-10 and natural (+)-1; b) synthetic (ꢀ)-10; c) synthetic (ꢀ)-1;
d) 1:2 mol ratio of synthetic (ꢀ)-1 and natural (+)-1. Concentrations
of approximately 10 mm, no solvent suppression.
Scheme 1. Synthesis of (ꢀ)-10. Reagents and conditions: a) TBDPSCl,
imidazole, DMF, 08C!RT, 4 h, 91%; b) MeOCH2Cl, Hünig’s base,
CH2Cl2, 08C!RT, 56 h, 98%; c) TBAF, THF, ꢀ108C, 4 h, 95%;
d) Lindlar cat., H2, (1 atm), EtOH, 14 h, 98%; e) BnBr, K2CO3, CH3CN,
31 h, 91%; f) 1. (COCl)2, DMSO, CH2Cl2, ꢀ788C; 2. Et3N, 94%;
g) 1. 5, nBu2BOTf, Et3N, CH2Cl2, ꢀ78!08C, 3 h; 2. 4, ꢀ78!08C,
2.5 h, 77%, d.r. 24:1; h) H2O2, LiOH, 08C, 30 min, 96%; i) TFA, 08C,
1 h, 98%; j) 1. 6, EDCI, HOBt, DMF, 08C, 10 min; 2. (ꢀ)-8, Et3N,
08C!RT, 1 h, 81%; k) 1. HCl, MeOH, H2 (5 atm), Pd/C, 1 h; 2. HCl,
H2O, H2 (5 atm), Pd/C, 1 h, 76%. Bn=benzyl, Boc=tert-butoxycar-
bonyl, DMF=N,N-dimethylformamide, DMSO=dimethyl sulfoxide,
EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
HOBt=1-hydroxybenzotriazole, MOM=methoxymethyl, TBAF=tetra-
n-butylammonium fluoride, TBDPS=tert-butyldiphenylsilyl, TFA=tri-
fluoroacetic acid.
Scheme 2. Synthesis of model (ꢀ)-1. Reagents and conditions:
a) 1. mW, toluene, 1108C, 15 min; 2. THF, NH3, 30 min; 3. NH3 (2m),
MeOH, 5 h; 4. NaOH (1n), MeOH, 4.5 h, 62%; b) TFA, 08C, 1 h,
99%; c) 1. EDCI, HOBt, DMF, 08C, 10 min; 2. (+)-8, Et3N, 08C!RT,
1 h, 88%; d) 1. HCl, MeOH, H2 (5 atm), Pd/C, 1 h; 2. HCl, H2O, H2
(5 atm), Pd/C, 1 h, 75%.
rearrangement and subsequent ammoniolysis). Removal of
the protecting groups of 12 under the reaction conditions that
were previously used in Scheme 1[14] gave (ꢀ)-1 in 75% yield.
The NMR spectra ofsynthetic ( ꢀ)-1 and natural (+)-1
were identical in all respects; co-addition ofnatural ( +)-1 to
topicities and 13C NMR chemical shift differences of a C2-
symmetric diamino tetraol derived from 2.[5] Considering that
the configuration of the amino acid in (+)-1 was unequiv-
1
1
ocally l,[5] the H and 13C NMR signals at C10–C15 showed
(ꢀ)-1 gave a single discrete set of H (Figure 2 d) and 13C
1
negligible differences, and the largest H NMR difference in
signals corresponding to those ofnatural ( +)-zwittermi-
cin A.[1a]
chemical shift occurred at H8, we hypothesized that the
mismatch resulted from the inversion of all configurations in
the diaminopolyol–carboxylate moiety of( ꢀ)-10: C8–C11,
C13, and C14. The latter (inversion ofconfiguration at C14)
would negate the original assumption ofa formal biosynthesis
of 1 derived from an l-Ser starter unit[4a,5] in the NRPS
loading domain and would therefore require the involvement
of d-Ser. To test this hypothesis compound 12, a diastereomer
of 9, was prepared by coupling carboxylic acid 6 with d-a-
aminoamide (+)-8 (88%; Scheme 2, where (+)-8 was derived
from the known acyl azide 11[16] in 2 steps by Curtius
Finally, the specific rotation of synthetic (ꢀ)-1 ([a]D =
ꢀ7.98, H2O) was opposite in sign and equal in magnitude to
natural zwittermicin A. Therefore the configuration of zwit-
termicin A ((+)-1) is 4S,8R,9S,10S,11S,13S,14R as depicted
(Figure 1). The assignment ofconifguration described here
has implications for the biosynthesis of (+)-1. Three scenarios
can be considered to explain the unexpected 14R configura-
tion ofzwittermicin A; 1) direct incorporation of d-Ser at
C13–C15 (path a); Scheme 3), which is similar to that
[17a]
observed for the d-Ala starter residue ofcyclosporine,
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Communications
related to non-specific interactions with the diaminopolyol
unit, but is more closely allied to either transport across the
cell wall or membrane, or a mechanism that implicates a more
subtle chiral recognition motifat an as-yet unidentiefd
intracellular target.
In summary, the absolute stereostructure of( +)-zwitter-
micin A ((+)-1) has been unambiguously assigned by total
synthesis of( ꢀ)-1 in an overall yield of1.9% (20 steps from
N,N-dibenzyl-l-serine methyl ester). Interpretation ofthe
configuration of (+)-1 implicates a “d-Ser” motifin the
biosynthesis ofC13–C15, which is consistent with an anti-
podal configuration of the propagated Ser starter unit.
Zwittermicin A and its enantiomer exhibit a pattern of
differential activity against fungal pathogens that underscores
the importance ofchirality to the biological activity ofthese
acyclic diaminopolyol natural products.
Scheme 3. Possible pathways for the propogation of d-Ser in the
biosynthesis of (+)-1). a) Loading of l-Ser and inversion at C14 of l-
Ser thioester catalyzed by an epimerase domain. b) Loading of l-Ser
and condensation with malonyl thioester with concomitant epimeriza-
tion. c) Loading of d-Ser.
2) a epimerization ofa carrier-protein-bound l-Ser by an
embedded epimerization domain (path b); Scheme 3), or
3) the involvement ofa dual-function condensation/epimeri-
zation domain (path c); Scheme 3), such as those operating in
the biosynthesis ofarthrofactin [17b] and enduracidin.[17c] In the
latter case, a single catalytic domain may be responsible for
inversion ofthe a configuration and coupling of the resultant
thioacyl d-Ser residue with a downstream acceptor residue,
however, this mechanism is yet to be associated with a mixed
NRPS/PKS system. Although details have been reported for
gene products ZmAG-ZmAI, which are responsible for the
AM extender unit,[4c] the identification of the genes and a
mechanism responsible for the Ser loading domain and its
incorporation into C13–C15 of 1 are still unclear. Resolution
ofthis mystery awaits more detailed annotation ofthe gene
cluster involved in the biosynthesis of( +)-1.
We have briefly compared the biological activity of (+)-
zwittermicin A with that ofits synthetic enantiomer ( ꢀ)-1, by
measuring the susceptibility of pathogenic fungi and flucona-
zole-resistant pathogens ofthe genus Candida (Table 1).
The minimum inhibitory activities ofauthentic natural
(+)-1 against Candida albicans ATCC14503 (MIC =
55.7 mgmLꢀ1) and the fluconazole-resistant strain C. albicans
96-489 (MIC = 59.5 mgmLꢀ1) were found to be comparable to
the antifungal activities found by Handelsman et al.[2a] for
(+)-1 against a range ofplant pathogenic fungi ofagricultural
importance. On the other hand, (ꢀ)-zwittermicin A was
inactive (MIC > 128 mgmLꢀ1) under the same conditions.
This interesting result implies that the activity of( +)-1 is not
Received: April 3, 2008
Published online: September 16, 2008
Keywords: amino alcohols · configuration determination ·
.
natural products · polyketides · total synthesis
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(ꢀ)-1 MIC
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C. albicans 96-489[b]
C. glabrata
55.7
59.5
>128
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>128
>128
>128
>128
>128
>128
C. albicans UCDFR1[c]
C. albicans ATCC14053
C. krusei
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