Bio-and air-tolerant carbon-carbon bond formations via organometallic ruthenium catalysis

Article abstract of DOI:10.1135/cccc2009053

Selected [2+2+2] cycloadditions, alkene-alkyne coupling and fusion of enyne with diazo compound, all triggered by an artificial organometallic ruthenium catalyst are demonstrated to proceed under ambient aerobic aqueous conditions in presence of bodily fl

Full text of DOI:10.1135/cccc2009053

Bio- and Air-Tolerant Carbon–Carbon Bond Formations
1023
BIO- AND AIR-TOLERANT CARBON–CARBON BOND FORMATIONS
VIA ORGANOMETALLIC RUTHENIUM CATALYSIS
1
2
3
4
5
Louis ADRIAENSSENS , Lukáš SEVERA , Jan VÁVRA , Tereza ŠÁLOVÁ , Jakub HÝVL ,
6
7
8
9,
Martina ČÍŽKOVÁ , Radek POHL , David ŠAMAN and Filip TEPLÝ *
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: adriaenssens@uochb.cas.cz,
2
6
3
4
5
severa@uochb.cas.cz, vavra@uochb.cas.cz, salova.tereza@seznam.cz, hyvl@uochb.cas.cz,
7
8
9
cizkova@uochb.cas.cz, pohl@uochb.cas.cz, saman@uochb.cas.cz, teply@uochb.cas.cz
Received April 17, 2009
Accepted April 22, 2009
Publish ed on lin e Jun e 26, 2009
Dedicated to Dr. Alfred Bader on the occasion of his 85th birthday in recognition of his outstanding
contributions as a chemist, enterpreneur, and benefactor.
Selected [2+2+2] cycloaddition s, alken e–alkyn e couplin g an d fusion of en yn e with diazo
com poun d, all triggered by an artificial organ om etallic ruth en ium catalyst are dem on strated
to proceed un der am bien t aerobic aqueous con dition s in presen ce of bodily fluids or cell
lysate. To th e best of our kn owledge, th ese are th e first exam ples of bio- an d air-toleran t
C–C bon d form ation catalyzed by an artificial organ om etallic com poun d.
Keyw ord s: Ruth en ium ; Organ om etallic catalysis; C–C couplin g; [2+2+2] Cycloaddition s;
Alken e–alkyn e couplin g; En yn e-diazocom poun d fusion ; Alkyn es; Aren es; Heterocycles;
Nitrogen -based organ ic cation s; N-h eteroarom atic cation s; Helical structures; Helquat; Bio-,
air-, an d water-toleran t tran sform ation s; E. coli cell lysate; Hum an serum ; Bioorth ogon al re-
action s; Non -coded biocom patible ch em istry.
Con struction of carbon –carbon (C–C) bon ds is th e essen ce of organ ic
ch em istry1. In th is respect, catalysis by organ om etallics (com poun ds with
carbon –m etal bon ds) h as becom e a powerful tool2. However, catalytic m an -
ifolds in volvin g C–M bon ds are widely viewed as in com patible with water,
air, th iols, an d organ ic electroph iles3–5. Con sequen tly, th e gen eral lack of
robust catalytic processes supported un der aerobic aqueous ph ysiological
con dition s represen ts a fun dam en tal obstacle in th e application of organ o-
m etallic catalysis towards in situ syn th esis in ch em ical biology, diagn ostics,
an d th erapeutics. Referrin g to th is seldom discussed syn th etic aspect of
bioorgan om etallic ch em istry6, n otable exam ples of C–H (ref.7), C–N (ref.8),
an d C–S (ref.6) bon d form ation s catalyzed by organ om etallics un der aerobic
Collect. Czech. Chem. Commun. 2009, Vol. 74, Nos. 7–8, pp. 1023–1034
© 2009 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2009053

1024
Adriaenssens et al.:
con dition s in com plex cellular m edia h ave been reported. In th is vein , th e
iden tification of organ om etallic processes th at form C–C bon ds un der
ph ysiological con dition s represen ts a particularly im portan t ch allen ge9–11
.
RESULTS AND DISCUSSION
Here, we sh ow th at C–C bon d form ation can be perform ed usin g an artifi-
cial ruth en ium organ om etallic catalyst in presen ce of aqueous aerobic m e-
dia such as bodily fluids or cell lysate un der m ild con dition s. Recen tly,
Meggers an d Streu effected th e un cagin g of am in es from th e correspon din g
allylcarbam ates in livin g cells usin g com m ercially available catalyst 1
(ref.6). In oth er work directed towards organ ic syn th esis in bio-m edia, th e
groups of Bertozzi an d Sh arpless h ave sh own alkyn e fun ction ality to be sig-
n ifican tly biorth ogon al12,13. In spired by th ese fin din gs, we speculated th at
bio-toleran t C–C bon d form in g processes could be foun d am on g th e rich
set of kn own alkyn e-based tran sform ation s catalyzed by 1 (ref.14). We ob-
served th at [2+2+2] cycloaddition of dim eth yl acetylen edicarboxylate (2)
using catalyst 1 (1 m ole %)15 was n ot on ly air-toleran t16 but also proceeded
in presen ce of water with out an y added organ ic co-solven t (Sch em e 1,
CO2Me
CO2Me
catalyst 1
MeO2C
MeO2C
CO2Me
Ru
Cl
medium
air, r.t.
CO2Me
CO2Me
CO2Me
Catalyst, 1
2
3
SCHEME 1
TABLE I
Bio- an d air-toleran t [2+2+2] cycloaddition of alkyn e 2a
En try
Catalyst 1, %
Tim e, h
Medium
Yield, %b
1
2
3
4
5
6
1
5
5
0
5
5
5.5
25
4
H2O
76c
72
Rattus norveg. urin ed
E. coli cell lysate
E. coli cell lysate
fetal bovin e serum
h um an serum f
76
0c,e
20
22
24
59
52
a
b
c
Medium (1 m l), 2 (100 µl, 0.81 m m ol). Isolated yields of 3. 2 m l of m edium used.
d
e
f
From Rattus norvegicus adult m ales. Startin g m aterial 2 was recovered in 56% yield. From
m ale, age 31.
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Bio- and Air-Tolerant Carbon–Carbon Bond Formations
1025
Table I, en try 1)17. Un der th ese m ild aerobic con dition s, th e startin g m ate-
rial was con sum ed with in 5.5 h an d h exam eth yl m ellitate 3 was isolated in
76% yield. Notably, th e reaction progressed to give sim ilarly good isolated
yields wh en perform ed with urin e of Rattus norvegicus, cell lysate from Esch-
erichia coli, fetal bovin e serum , an d h um an serum (en tries 2–6). To th e best
of our kn owledge, th is is th e first exam ple of C–C bon d form ation catalyzed
by an artificial organ om etallic com poun d in th e presen ce of air an d bodily
fluids or cell lysate.
Th e study was exten ded to in clude [2+2+2] cycloaddition affordin g
pyridin e derivative 6 (Sch em e 2)16a,18,19, an d Alder-en e-type couplin g of
alkyn e 7 with allyl alcoh ol (8)16a,20,21, to give a m ixture of aldeh ydes 9 an d
10 (Sch em e 3). Of n ote is th at aldeh yde fun ction ality is gen erated in th is
process. Such a feature m igh t be furth er explored in th e con text of th e fol-
low-up reactivity profile of aldeh ydes in livin g system s22. Product 9 is pres-
en t as a m ixture of th e open ch ain aldeh yde 9a an d its cyclic
seven -m em bered h em iacetal isom er 9b (9a:9b 75:25). In terestin gly, syn th e-
sis of 9 th us represen ts organ om etallic assem bly of a carbon fram ework
rem in iscen t of aldoh eptose in a bio-relevan t en viron m en t in situ.
CN
CN
1 (5 mole %)
CN
O
O
+
N
fetal bovine serum
air, r.t., 3 h
4 (0.60 M)
5
57%
6
SCHEME 2
OH
O
OH
HO
O
1 (5 mole %)
OH
+
9a
9b
10
E. coli cell lysate
+
air, r.t., 15 min
OH 7 (0.53 M)
8
O
58 % (9:10 7:3)
SCHEME 3
An exam ple of en yn e-diazo com poun d fusion to give bicyclic cyclopro-
pan es 13 an d 14 furth er expan ds th e scope of bio-toleran t tran sform ation s
catalyzed by 1 (Sch em e 4)16a,23,24. Th is tran sform ation proceeds rem arkably
fast with all th e m edia tested (E. coli cell lysate, com bin ed yield of 13 an d
14 is 60%; Rattus norvegicus urin e, 57%; h um an serum , 45%).
Heterogen eous catalysis an d tran sform ation s takin g place ‘on water’ 5c
can n ot be excluded as substrates, products an d also th e catalyst 1 in exam -
ples in Table I an d Sch em es 2–4 were partly in soluble un der th e reaction
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1026
Adriaenssens et al.:
con dition s presen ted. Th erefore, we n ext turn ed to a tran sform ation wh ere
better h om ogen eity of th e reactin g m ixture is en sured. Specifically, we ex-
am in ed th e feasibility of syn th esis of our n ovel water-soluble h elical ex-
ten ded diquat (h elquat)25 in E. coli cell lysate with an added 1% aceton e to
aid solubilization of th e catalyst (Sch em e 5).
TMS
1 (6 mole %)
TMS
Ts-N
Ts-N
Ts-N
+
TMS
N2
+
fetal bovine serum
air, r.t., 5 min
13
14
64% (13:14 5:1)
11 (0.17 M)
12
SCHEME 4
2X-
2X-
N+
N+
N+
1 (25 mole %)
1% Me2CO in E. coli cell lysate
air, 37 °C, 36 h
N+
51 %
X = CF3SO3
16
15 (12 mM)
SCHEME 5
As judged by NMR an alysis of th e evaporated reaction m ixture, progres-
sive disappearan ce of th e peaks correspon din g to triyn e 15 an d em ergen ce
of peaks diagn ostic of h elquat 16 dem on strates th e feasibility of th is pro-
cess un der aerobic bio-relevan t con dition s (Fig. 1). In crease in yield from
33% after 2.5 h to 51% after 36 h , dem on strates th at th e (C5Me5)Ru-based
organ om etallic catalyst stays active for exten ded periods in th is m edium 26
.
Notably, th e reaction proceeds effectively even in th e presen ce of con sid-
erable am oun ts of bovin e serum album in , a protein kn own for its prom is-
cuous bin din g profile (Fig. 2)27.
Poten tially, a reaction proceedin g via organ om etallic in term ediates m igh t
be viewed as an en try to a collection of species with poten tial bioactivity
(Fig. 3). Th e possibility of search in g th rough such species gen erated in situ
in presen ce of biological targets con tain ed in th e reaction m edium , m igh t
allow for in vestigation of bin din g or reactive even ts oth erwise difficult or
even im possible to realize. Such an approach seem s particularly attractive
in ligh t of th e fact th at (C5H5)Ru an d oth er Ru-aren e organ om etallics h ave
been sh own to be poten t an d selective bioactive en tities28.
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Bio- and Air-Tolerant Carbon–Carbon Bond Formations
1027
FIG. 1
1H NMR (400 MHz, D2O) m on itored progression of Ru-catalyzed cycloaddition of 15 in E. coli
cell lysate (Sch em e 5). From solvation of startin g m aterial 15 in E. coli cell lysate before addi-
tion of catalyst 1 (top pan el) to en d of reaction after 36 h as judged by disappearan ce of 15
(bottom pan el)
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1028
Adriaenssens et al.:
In sum m ary, we h ave presen ted eviden ce th at form ation of C–C bon ds
via ruth en ium organ om etallic catalysis can be ach ieved in th e presen ce of
bio-relevan t m edia such as fetal bovin e serum or E. coli cell lysate un der
aerobic am bien t con dition s. Th is will be of con ceptual an d practical in ter-
est for organ om etallic carbon fram ework con struction targeted towards
bio-application s in cludin g ch em ical biology, diagn ostics, an d th erapeutics.
FIG. 2
1H NMR (400 MHz, D2O) of reaction m ixture from tran sform ation 15 → 16 in th e presen ce of
bovin e serum album in an d E. coli cell lysate
FIG. 3
An un -n atural organ om etallic reaction in bio-relevan t m edia m igh t be viewed as an en try to
a collection of species with poten tial bioactivity
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Bio- and Air-Tolerant Carbon–Carbon Bond Formations
1029
EXPERIMENTAL
Unless otherwise noted, all reactions were carried out under aerobic conditions with no efforts taken
to exclude air and moisture. Liquids an d solution s were added via n eedle an d syrin ge un less
oth erwise stated. Th in -layer ch rom atograph y (TLC) an alysis was perform ed on silica gel
plates (Silica gel 60 F254-coated alum in ium sh eets, Merck, cat. No. 1.05554.0001) an d visual-
ized by UV (UV lam p 254/365 n m , Spectrolin e Model ENF – 240C/FE) an d, eith er ch em ical
stain in g with KMn O4 [KMn O4 (1% aq.), Na2CO3 (2% aq.)], or Ce(SO4)2 [Ce(SO4)2·4H2O
(1%), H3P(Mo3O10
) (2%) in sulfuric acid (10% aq.)]. TLC an alysis of dication s was ach ieved
4
usin g Stoddart’s m agic m ixture29 (MeOH:NH4Claq(2 M):MeNO2 70:20:10) as eluen t on silica
gel plates. Flash ch rom atograph y was perform ed on silica gel 60 (Fluka, cat. No. 60741) with
th e in dicated eluen t. All dilution s perform ed to determ in e th e yield of 16 were perform ed
with volum etric glassware an d/or Ham ilton Microliter syrin ges.
Materials
Un less oth erwise stated, all startin g m aterials an d reagen ts were obtain ed from com m ercial
suppliers an d used with out furth er purification . Dem in eralized water obtain ed from th e
Water Purification Facility at th e In stitute of Organ ic Ch em istry an d Bioch em istry, Academ y
of Scien ces of th e Czech Republic was used un less oth erwise stated. Dem in eralization was
accom plish ed via filtration th rough ion exch an ge colum n s (Lewatit S100 for catex colum n ,
Lewatit MP500 for an ex colum n ) in a dem in eralization ion exch an ge station type ID-PP an d
ID-KP (Kavalier, Votice, Czech Republic).
Rattus norvegicus urine. Th e urin e from several m ale rats (Rattus norvegicus species, Wistar,
supplier: Bio-Test s.r.o. Kon árovice, Czech Republic) was pooled an d kept frozen (–20 °C) un -
til furth er use. Before an experim en t, it was th awed in a 30 °C water bath .
Escherichia coli cell lysate. Escherichia coli DH5α tran sform ed with plasm id pET24a
(Novagen , cat. No. 69749-3) were grown in LB broth (Sigm a, L3022) con tain in g kan am ycin
(40 µg/m l, Fluka 60615). 40 m l of LB m edia were in oculated with 4 µl of saturated culture
an d grown overn igh t at 37 °C an d 220 rpm . Th e culture was spun down at 3500g for 15 m in
an d th e pellet was resuspen ded in 10 m l of ph osph ate buffered salin e (PBS) an d son icated
(6 × 1 m in at 3-m in in tervals). Th e crude lysate was th en spun down at 10000g for 30 m in
at 4 °C an d th e supern atan t was decan ted. Th e solution was stored at –20 °C. Before an
experim en t, it was th awed in a 30 °C water bath . Th e residue con ten t of th e m aterial was
35 m g/m l.
Fetal bovine serum (FBS). FBS was purch ased from In vitrogen -Gibco (cat. No. 10270-106).
Before use, FBS was h eated at 65 °C for 30 m in to in activate th e com plem en t system . Th e
residue con ten t of th e m aterial was 30 m g/m l.
Human serum. On e of th e auth ors (m ale, 31) don ated th e blood (B n egative blood type).
All blood an alysis data were with in n orm al ran ge an d HIV 1 + 2 n egative. Th e serum was
obtain ed by cen trifugation of th e wh ole blood. Serum was stored in th e freezer (–20 °C) an d
th awed before use in a 30 °C bath . Th e residue con ten t of th e m aterial was 100 m g/m l.
Hexam eth yl Ben zen e-1,2,3,4,5,6-h exacarboxylate (3). Gen eral Procedure
Dim eth yl acetylen edicarboxylate 2 was added dropwise via Ham ilton syrin ge un der open
flask con dition s to a suspen sion of [Cp*Ru(cod)Cl] 1 in th e described m edium . Th e reaction
flask was closed with a stopper to preven t evaporation an d th e reaction m ixture stirred for
Collect. Czech. Chem. Commun. 2009, Vol. 74, Nos. 7–8, pp. 1023–1034

1030
Adriaenssens et al.:
th e described tim e at room tem perature (r.t.). Form ation of th e product an d con sum ption of
th e startin g m aterial was detected by TLC (petroleum eth er:eth yl acetate 50:50, product
RF 0.29, startin g m aterial RF 0.85). Th e reaction m ixture was diluted with water an d ex-
tracted with eth yl acetate (5 ×). Th e com bin ed organ ic ph ases were dried over an h ydrous
Na2SO4. Th e m ixture was filtered an d th e solven t was rem oved in vacuo to give crude prod-
uct wh ich was purified by flash ch rom atograph y on silica gel (petroleum eth er:eth yl acetate
60:40). Hexam eth yl ben zen e-1,2,3,4,5,6-h exacarboxylate 3 was obtain ed as colorless crystals.
Th e spectroscopic ch aracterization data are in agreem en t with th e literature17
.
Table I, entry 1. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), [Cp*Ru(cod)Cl] 1 (3.5 m g, 9.2 µm ol, 1 m ole %), water (2 m l) as th e m edium , an d
a reaction tim e of 5.5 h , h exam eth yl ben zen e-1,2,3,4,5,6-h exacarboxylate 3 was obtain ed as
colorless crystals in 76% yield (88.3 m g, 0.207 m m ol).
Table I, entry 2. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), [Cp*Ru(cod)Cl] 1 (15.5 m g, 40.8 µm ol, 5 m ole %), urin e of Rattus norvegicus (1 m l)
as th e m edium , an d
a reaction tim e of 25 h , h exam eth yl ben zen e-1,2,3,4,5,6-h exa-
carboxylate 3 was obtain ed as colorless crystals in 72% yield (83.3 m g, 0.195 m m ol).
Table I, entry 3. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), [Cp*Ru(cod)Cl] 1 (15.5 m g, 40.8 µm ol, 5 m ole %), E. coli DH5α cell lysate (1 m l) as
th e m edium , an d a reaction tim e of 4 h , h exam eth yl ben zen e-1,2,3,4,5,6-h exacarboxylate 3
was obtain ed as colorless crystals in 76% yield (87.3 m g, 0.205 m m ol).
Table I, entry 4. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), E. coli DH5α cell lysate (2 m l) as th e m edium , an d a reaction tim e of 20 h , but
om ittin g [Cp*Ru(cod)Cl] 1 (0 m g, 0 µm ol, 0 m ole %), th e startin g m aterial dim eth yl
acetylen edicarboxylate 2 was obtain ed as an oil in 72% yield (83.3 m g, 0.195 m m ol). Th e
spectroscopic ch aracterization data are in agreem en t with th at of th e com m ercial sam ple.
Table I, entry 5. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), [Cp*Ru(cod)Cl] 1 (15.5 m g, 40.8 µm ol, 5 m ole %), fetal bovin e serum (1 m l) as th e
m edium , an d a reaction tim e of 22 h , h exam eth yl ben zen e-1,2,3,4,5,6-h exacarboxylate 3
was obtain ed as colorless crystals in 59% yield (68.6 m g, 0.161 m m ol).
Table I, entry 6. Usin g dim eth yl acetylen edicarboxylate 2 (100 µl, 115.6 m g, 0.813 m m ol,
1 equiv.), [Cp*Ru(cod)Cl] 1 (15.6 m g, 41.1 µm ol, 5 m ole %), h um an serum (1 m l) as th e m e-
dium , an d a reaction tim e of 24 h , h exam eth yl ben zen e-1,2,3,4,5,6-h exacarboxylate 3 was
obtain ed as colorless crystals in 52% yield (60.2 m g, 0.141 m m ol).
2-(1,3-Dih ydrofuro[3,4-c]pyridin -6-yl)aceton itrile (6)
Propargyl eth er 4 (62 µl, 56.7 m g, 0.602 m m ol, 1 equiv.) was added dropwise over 15 m in
via Ham ilton syrin ge to a stirrin g suspen sion of m alon on itrile 5 (61.6 m g, 0.932 m m ol,
1.5 equiv.) an d [Cp*Ru(cod)Cl] 1 (11.4 m g, 30 µm ol, 5 m ole %) in fetal bovin e serum (1 m l).
Th e reaction m ixture was th en stirred at r.t. for 3 h . Form ation of th e product was detected
by TLC (h exan es:eth yl acetate 50:50, RF 0.18). Th e reaction m ixture was diluted with water
an d extracted with DCM (1 ×) an d eth yl acetate (3 ×). Th e organ ic fraction s were com bin ed
an d dried over an h ydrous Na2SO4. Th e m ixture was filtered an d con cen trated in vacuo to
yield th e crude product wh ich was purified by flash ch rom atograph y on silica gel (h exan es:
eth yl acetate 40:60). 2-(1,3-Dih ydrofuro[3,4-c]pyridin -6-yl)aceton itrile 6 was obtain ed as a
colorless am orph ous solid in 57% yield (55.0 m g, 0.343 m m ol). Th e spectroscopic ch aracter-
ization data are in agreem en t with th e literature18
.
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Bio- and Air-Tolerant Carbon–Carbon Bond Formations
1031
6-Hydroxy-4-m eth ylen eh exan al (9) an d (E)-7-Hydroxyh ept-4-en al (10)
Alkyn e 7 (40 µl, 37.1 m g, 0.529 m m ol, 1 equiv.) was added dropwise via Ham ilton syrin ge
un der open flask con dition s to a stirrin g suspen sion of allyl alcoh ol 8 (110 µl, 93.8 m g,
1.614 m m ol, 3.1 equiv.) an d [Cp*Ru(cod)Cl] 1 (10.3 m g, 0.0271 m m ol, 5 m ole %) in E. coli
DH5α cell lysate (1 m l). Th e reaction m ixture was th en stirred at r.t. for 15 m in . Form ation
of two products was detected by TLC (n-pen tan e:dieth yl eth er 50:50, product 9, RF 0.23;
product 10, RF 0.10). Th e reaction m ixture was diluted with water an d extracted with
dieth yl eth er (3 ×). Th e organ ic fraction s were com bin ed an d dried over an h ydrous Na2SO4.
Th e m ixture was filtered an d con cen trated in vacuo to give th e crude product wh ich was
purified by flash ch rom atograph y on silica gel (n-pen tan e:dieth yl eth er 50:50). A 68:32 ratio
of isom eric products 9 an d 10 was obtain ed as a yellowish oil in 58% yield (39.3 m g,
0.307 m m ol). Repeated ch rom atograph y allowed sam ples of each isom er to be isolated (9 as
a 76:24 ratio of th e open ch ain an d cyclic h em iacetal isom ers 9a an d 9b). Th e spectroscopic
ch aracterization data are in agreem en t with th e literature20
.
(Z)-3-Tosyl-1-(2-(trim eth ylsilyl)vin yl)-3-azabicyclo[3.1.0]h exan e (13) an d
(E)-3-Tosyl-1-(2-(trim eth ylsilyl)vin yl)-3-azabicyclo[3.1.0]h exan e (14)
(Trim eth ylsilyl)diazom eth an e 12 (110 µl, 2 M solution in dieth yl eth er, 0.220 m m ol, 1.2 equiv.)
an d [Cp*Ru(cod)Cl] 1 (4 m g, 10.5 µm ol, 6 m ole %) were successively added with in 1 m in
un der open flask con dition s to a stirrin g suspen sion of en yn e 7 (45.8 m g, 0.1836 m m ol,
1 equiv.)30 in fetal bovin e serum (1 m l). Th e reaction m ixture was th en stirred vigorously at
r.t. for 5 m in . Form ation of th e products was detected by TLC (h exan es:eth yl acetate 80:20,
product RF 0.57). Th e reaction m ixture was diluted with water an d extracted with dieth yl
eth er (3 ×). Th e organ ic fraction s were com bin ed an d dried over an h ydrous Na2SO4. Th e
m ixture was filtered an d con cen trated in vacuo to yield th e crude product wh ich was puri-
fied by flash ch rom atograph y on silica gel (h exan es:eth yl acetate 15:1, RF 0.20). An in -
separable 5:1 m ixture of isom ers 13 an d 14 was obtain ed as a brown ish oil in 64% yield
(39.2 m g, 0.117 m m ol) an d a sm all am oun t of desilylated by-product was also isolated in
6% yield (2.7 m g, 0.010 m m ol). Th e spectroscopic ch aracterization data are in agreem en t
with th e literature23,31
.
6,7,12,13-Tetrah ydro-5,14-diaza[5]h elicin ium Trifluorom eth an esulfon ate (16)
Procedure to demonstrate reaction development in transformation 15 → 16. On e drop of
aceton e (0.01 m l) was added directly to th e bottom of a RBF con tain in g [Cp*Ru(cod)Cl] 1
(1.7 m g, 4.48 µm ol, 25 m ole %). A solution of 2,2′-(eth yn e-1,2-diyl)bis(1-(but-3-yn yl)-
pyridin ium )bis(trifluorom eth an esulfon ate) 15 (10.6 m g, 18.1 µm ol, 1 equiv.)25 in E. coli
DH5α cell lysate (1.5 m l) was added to th e m ixture of catalyst an d aceton e. Th e resultin g so-
lution was stirred at 37 °C. Aliquots (0.3 m l) of th e reaction m ixture were rem oved at reac-
tion tim es of 2.5, 5, 24, an d 36 h . Th ese aliquots were con cen trated in vacuo an d an alyzed
by 1H NMR (Fig. 1).
Procedure for yield determination in transformation 15 → 16. On e drop of aceton e (0.01 m l)
was added directly to th e bottom of a RBF con tain in g [Cp*Ru(cod)Cl] 1 (1.6 m g, 4.28 µm ol,
25 m ole %). A solution of 2,2′-(eth yn e-1,2-diyl)bis(1-(but-3-yn yl)pyridin ium )bis(trifluoro-
m eth an esulfon ate) 15 (10.0 m g, 17.1 µm ol, 1 equiv.)25 in E. coli DH5α cell lysate (1.5 m l)
was added to th e m ixture of catalyst an d aceton e. Th e resultin g solution was stirred at 37 °C
Collect. Czech. Chem. Commun. 2009, Vol. 74, Nos. 7–8, pp. 1023–1034

1032
Adriaenssens et al.:
for 36 h . Th e yield of th is reaction was determ in ed by 1H NMR with com parison to DMSO
as an in tern al stan dard.
Procedure for transformation 15 → 16 with added bovine serum albumin. On e drop of aceton e
(0.01 m l) was added directly to th e bottom of a RBF con tain in g [Cp*Ru(cod)Cl] 1 (0.8 m g,
2.14 µm ol, 25 m ole %). A solution of 2,2′-(eth yn e-1,2-diyl)bis(1-(but-3-yn yl)pyridin ium )-
bis(trifluorom eth an esulfon ate) 15 (5.0 m g, 8.6 µm ol, 1 equiv.)25 an d bovin e serum album in
(26.4 m g) in E. coli DH5α cell lysate (1 m l) was added to th e m ixture of catalyst an d ace-
ton e. Th e resultin g solution was stirred at 37 °C for 36 h . Th e reaction m ixture was con cen -
trated in vacuo. 1H NMR an alysis in dicates form ation of 6,7,12,13-tetrah ydro-5,14-diaza-
[5]h elicin ium trifluorom eth an esulfon ate 16 an d con sum ption of th e startin g m aterial 15
(Fig. 2).
Supportin g In form ation Available
Furth er an alytical data, scan n ed NMR spectra, an d yield determ in ation for tran sform ation in
Sch em e 5 are available free of ch arge at h ttp://dx.doi.org/10.1135/cccc2009053.
Support of this work by the Institute of Organic Chemistry and Biochemistry, Academy of Sciences
of the Czech Republic, v.v.i. (Z4 055 0506 and IOCB Postdoctoral Fellowship to L.A.) and Czech
Science Foundation (203/09/0705 and 203/09/1614) is gratefully acknowledged. W e thank J.
Konvalinka, I. Starý and D. Schröder for stimulating discussions, and M. Kožíšek, L. Machala, J.
Sedláková and J. Slaninová for experimental help and support.
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Adriaenssens et al.:
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