R e p r i n t e d f r o m t h e . l o u r n a l o f t h e A m e r i c a n C h e m i c a l S o c i e t v , 1 9 8 1 ,1 ( / J .4 8 9 0 .
O o p y r i g h t O 1 9 8 1 b y t h e A m e r i c a n C h e m i c a l S o c i e t l -a n d r e p r i n t e d b . " -p e r m i s s i o n o 1 ' t h ec o p v r i g h t o w n e r
Enzyme-Catalyzed
OrganicSynthesis:NAD(P)H Cofactor
Regeneration
by UsingGlucose6-Phosphate
and the
from Leuconostoc
Dehydrogenase
Glucose-6-phosphate
mesenteroidesr
Chi-Huey Wong and George M. Whitesides*
Contribution from the Department of Chemistry, MassachusettsInstitute of Technology,
Cambridge, Massachusetts 02139. ReceiuedDecember I I, 1980
(from Leuconostoc
Abstract Glucose-6-phosphate
dehydrogenase
comprisea useful
andglucose6-phosphate
mesenteroides)
systemfor regenerationof reducednicotinamidecofactorsfor use in enzyme-catalyzed
organicsynthesis.This enzymeis
stable,and easily
approximately
equallyactivein reductionof NAD and NADP. [t is commercially
available,inexpensive,
phosphorylation
immobilized.Glucose6-phosphate
of glucoseby ATP
can be preparedin quantity by hexokinase-catalyzed
(coupledwith ATP regeneration)
or by othermethods.The operationof this regeneration
systemis illustratedby syntheses
of enantiomerically
enrichedo-lacticacid (0.4 mol, enantiomericexcess95%)and (S)-benzyl-a-dlalcohol(0.4 mol, enantiomeric
e x c e s s 9 5 % ) a n d b y a s y n t h e s it shor ef o - o , ( * ) - i s o c i t r i c a c i d ( 0 . l 7 m oFla) .c t o r s i n f l u e n c i n g t h e s t a b i l i t y o f N A D ( P ) ( H ) i n
solutionhavebeenexplored.
Enzyme-catalyzed reactionsthat require reducednicotinamide
cofactors(NADH, NADPH) are not widely usedin preparative
chemistry.2'3Although many of thesercactionsare potentially
valuable in synthesis,the cofactorsrequiredare expensiveand
significantlyunstablein solutionand can be usedeconomically
in stoichiometricreactionsonly on a small scale. A number of
procedures for regenerating the reduced cofactors have been
proposedand tested.4 Of these,the use of formate and formate
d e h y d r o g e n a s(eE C 1 . 2 . 1 . 2 )t o r e d u c eN A D t o N A D H i s , i n
principle, one of the most practical,s'6although the enzyme used
in this procedure(from Candida boidinii) is still expensivewhen
purchasedcommercially. This formate dehydrogenase
doesnot
reduce NADP,7 although formate dehydrogenasefrom Clostri(l) Supportedby the National Institutesof Health, Grant GM 24025.
(2) Suckling,C. J. Chem.Sou.Reu.1974,384-407;
Suckling,C. J.; Wod,
H . C . S . C h e m .B r . 1 9 7 9 , 1 5 , 2 4 3 8 .
(3) Jones,J. B.; Beck,J, F. ln "Applicationsof BiochemicalSystemsin
O r g a n i cC h e m i s t r y " J; o n e sJ, . B . ; P e r l m a nO
, . ; S h i h ,C . J . , E d s . ;W i l e y - l n t e r s c i e n c e :N e w Y o r k , 1 9 7 6 ;p p 1 0 7 - 4 0 1 ;J o n e s ,J . B . I n " N e z y m i c a n d
N o n e n z y m i cC a t a l y s i s "D
, u n n i l l ,P . ; W i s e m a n A
, . ; B l a k e b r o u g hN, . , E d s . l
E l l i s H a r w o o dL t d . , 1 9 8 0 ;p p 5 8 - 8 3 .
(4) Wang, S. S.; King, C.-K. Adu. Biochem.Eng. 1919, 12, ll9 146:
Baricos,W.; Chambers,R.; Cohen,W.EnzymeTechnol.Dig. 1975,2,39-53.
( 5 ) S h a k e d ,Z . ; W h i t e s i d e sG, . M . J . A m . C h e m .S o c . 1 9 8 0 ,1 0 2 , 7 1 0 4 .
(6) Wichmann,R.; Wandry,C.; Biickmann,A. F.l Kula, M. R., Abstracts,
VIth InternationalFermentationSymposium,July 1980,London,Ontario,
( P ) .p 1 2 5 .
N a t i o n a lR e s e a r c C
h ouncilO
. t t a w a .C a n a d aA
: b s t r a c tF - 1 2 . 1 . 2 4
dium thermoacticum doesdo so.
This paper describesa regenerationsystem based on dehydrogenationof glucose6-phosphate(G-6-P) catalyzedby gludehydrogenase(G-6-PDH, G-6-P, NAD(P)
cose-6-phosphate
EC LI.I.49, from Leuconostocmesenteroides\.
oxidoreductase,
The G-6-P requiredcan be preparedby severalprocedures.The
most convenientfor synthesescarried out on a - 1 mol scaleis
phosphorylationof glucosewith ATP,
the hexokinase-catalyzed
with coupledacetatekinasecatalyzedregenerationof ATP by
using acetyl phosphate.The preparationsof acetyl phosphates
have been describedpreviously. For
and glucose6-phosphatee
larger scale work, routes based on conversionof starch and
phosphateto glucose6-phosphate(using phosphorylasea and
phosphoglucose
mutase) may also prove useful. The advantages
of the G-6-PHD from Leuconostocmesenteroidesas a catalyst
for usein organicsyntheticproceduresare that it is almostequally
effectivein reducingNAD and NADPTO'rrand that it is stable,
( 7 ) A n d r e e s e nJ,. R . ; L j u n g h d a h lL, . G . J . B a c t e r i o l . 1 9 7 41,2 0 , 6 - 1 4 . I n
principle,the NAD and NADP cofactorsystemscan be coupledby using
nicotinamide
n u c l e o t i d et r a n s - h y d r o g e n a(sEeC 1 . 6 . 1 . 1 . )C; o l o w i c k ,S . P . ;
K a p l a n ,N . O . ; N e u f e l d ,E . F . ; C i o t t i , M . M . J . B i o l . C h e m . 1 9 5 2 , 1 9 5 ,
95 105.
( 8 ) L e w i s ,J . M . ; H a y n i e ,S . L . l W h i t e s i d e sG, . M . " r . O r g . C h e m . 1 9 7 9 ,
44.864-5.
( 9 ) P o l l a kA
s ,. M . J . A m . C h e m .S o c . 1 9 7 7 ,
, . ; B a u g h nR
, . L.; WhitesideG
9 9 , 2 3 6 67 .
lVA D( P) H Cofactor Regeneration
J . A m . C h e m . S o c . ,V o l . 1 0 3 , N o . 1 6 , I 9 8 l
commerciallyavailable,and inexpensive.Thus, a single,practical,
experimentalprotocolcan be used interchangeablyto regenerate
both of the reduced nicotinamide cofactors. The latter feature
has beennoted by others,and this systemhas beenusedpreviously
in small-scaleexperiments.l2 Until recently,however,the cost
of G-6-P ( - $ 1800mol-t) has beensufficientlyhigh to discourage
its useas a stoichiometricreagentin synthesison the scaleof moles.
Here we establishthat the G-6-P required can be preparedconveniently and inexpensivelyin large quantities by severalmethods.
We illustrate the operation of this regenerationsystem by the
preparation of three chiral materials-o-lactic acid, threo-o"(+)-isocitric acid, and (S)-benzyl-a-d1alcoholr3-in 0. I -0.5 mol
scale.
Results
Stability of Nicotinamide Cofactors in Solutions. NAD(P)H.
In order to developpractical protocols for using and regenerating
the nicotinamidecofactors.it is useful to know the factors which
influence the lifetimes of these intrinsically unstable speciesin
solution. Qualitatively,the stability of NAD(P)H is greaterin
basic than in acidic solutions,and the stability of NAD(P) is
greater in acidic than in basicsolutions.ra Severalof the reactions
that transform NADH and NAD into enzymaticallyinactive
substanceshave been studied: in particular, NADH undergoes
acid-catalyzedhydration and, ultimately, transformationto a cyclic
ether;raNAD reactswith hydroxideion and with other nucleophiles.rs NAD is also subjectto enzyme-catalyzed
addition of
nucleophiles.l6 Analogous reactionsundoubtedlyoccur with
NADP(H) but have beenstudiedin lessdetail. Of thesereactions,
the most important in determiningthe stability of NAD(P)H
under the conditionsencounteredduring enzyme-catalyzedorganic
synthesisis the Lewis acid catalyzed hydrolysis reaction. This
processhas been studied in somedetail by Johnsonand Tuazon,
who have suggestedprotonationof C5 of the nicotinamidering
as rate limiting.'a Our studiesof this reactionparalleledthose
of these workers but have had a different and more practical
emphasis. Where the two studiescan be compared,agreement
is satisfactory.
The pH of a solution used in an enzyme-catalyzedsynthetic
reactionis usually fixed (+0.5 pH units) by the stabilitiesand
pH-rate profiles of the enzymesinvolved,and it is not practical
to adjust the stability of the cofactorsby changingthe solution
pH. Our interest in the subject of the solution stability of these
substances
was thereforecenteredon the influenceof buffers and
other solution componentsand especiallyon the influenceof
potential Lewis acid catalysts.
We have determined rates of disappearanceof NAD(P)H
spectrophotometrically.These rates follow eq I and 2. Figure
-d ln
[NADH]/dt = kNA'HoM =
/ c H [ H " ]+ k H A [ H A ] + k H , o ( l )
-d ln
[NADPH] /dt = ft^oor"o*o
- riNeo'ob'
=
knoorno*o
##;i
=
F#"-"
(2)
(3)
( 1 0 ) D e M o s s ,R . D . ; G u n s a l u sI,. C . ; B a r d .R . C . B a c t e r i o l .1 9 5 3 ,6 6 ,
"f.
l 0 - 1 6 ; L e v y , H . R . A d t t .E n z y m o l . 1 9 7 9 4, 8 , 9 7 1 9 2 .
( l l ) O t h e r s y s t e m st h a t a c c e p tb o t h N A D a n d N A D P ; g l u c o s ed e h y d r o g e n a sS
e ,t r c c k e r H
, . J . ; K o r k e s ,S . " / . B i o l . C h e m . 1 9 5 2 1
, 96,769-84;
6-PG dehydrogenase,
Frampton,E. W.; Wood, W. A. "r. Biol. Chem.1961,
236,2571-1;malatedehydrogenase,
Harry, l.; Korey.S. R.; Ochoa,S.J. Biol.
Chem. 1953,203, 594-604. All thcseenzvmesare expensive.
( 1 2 ) D u t l e r ,H . ; C o o n ,M . i . ; K u l l , A . ; V o g e l ,H . ; W a l d v o g e lG, . ; P e r l o g ,
Y. Eur. J. Biochem.1971,22.203-12.For an analytical-scale
determination
o f N A D P b y u s i n ge n z y m a t i c y c l i c ,s e e : C h i , M . M . Y . ; L o w r y ,C V . ; O .
H. Anal. Biochem.1978, 89, 119-29.
( I 3) Synthesisof (S)-benzyl-a-dlalcoholby reductionof benzaldehyde-a-d
w i t h 9 - B B N : M i d l a n d ,M . M . : G r e e r .S . ; T r a m o n t a n oA. . : Z d e r i c -S . A . " / .
A m . C h e m . S o c .1 9 7 9 ,1 0 1 , 2 3 5 2 - 5 5 .
( 1 4 ) J o h n s o nS, . L . ; T a u z o n P
, . T . B i o c h e m i s t r1y9 7 7 , 1 6 , 1 1 7 5 - 8 3 : O p p e n h e i m e rN, . J . ; K a p l a n ,N . O . I b i d . 1 9 7 4 . 1 3 , 4 6 1 5- 8 5 .
( 1 5 ) J o h n s o nS
, . L . ; M o r r i s o n ,D . L . B i o c h e m i s t r v1 9 7 0 , 9 , 1 4 6 0 7 0 .
( 1 6 ) E e v e r s eJ,. ; B a r n e t t ,R . E . ; T h o r n e ,C . J . R . ; K a p l a n ,N . O . l r c f r .
B i o c h i m .B i o p h y s . 1 9 7 11, 4 3 , 4 4 4 - 6 0 W
, i l t o n ,D . C . B i o c h e n .J . 1 9 7 9 , 1 7 7 .
9 5 1 - 7.
lso-.r
3.8_ -.-o
A
i
l7o--r
--,,.-o.-' ,
3'8
^
J'
,. o-''
I
I
I
'
r
.r'
lsoi.--.
4.q-a
4Oi
J
-ni
tt
v
a'9--t
,,-,o--
o
i',-',-'oos.a
C()v--r.o
J
-i-- lecac.a _
T-.
aa
:
*l
^
I
JI
-l-
r
4891
-l--l
I qOAc/OAc
i
I _-.'__--'I-
r-
T------r----
-i
I
6-
T
E
l
^)
I)
o'
6.O
--''
I
---46.5
-N
!.{' 2 pn
,/Frpni
'
"4/
9
o.o8 -[
L
o.o 6 _-.r
-..1
o.o 4 --1
a--t-
I
-1
ll'/T:
I
O.O2 ---l
I
o1,--r
,
o24
I
r
6
8
--'l
to
to'[Hx],u
Figurel. Catalysis
of disappearance
of NADH (O) andNADPH (O)
by acidsasa functionof IHX]. Valuesof pH usedarelistedon theplots.
ro
-'s
B
l
.
,,
-j
,/
(]--i
/
o
I
-l
o><
Or
J
ro
)
j
,/
I
468
ro7[H+] , n,t
(obtained
Figure2. A plot of k1Hyl=soM
by extrapolating
the linesof
F i g u r eI t o [ P i l = Q )v s .i H * 1 y i e l a s l o p e= 9 . 4 x 1 0 3M - r h | = f t x *
x 1 0a h ' ( * k H r o= 1 . 2 6x l 0 - { M ' h r ) .
a n di n t e r c e p=t O . ' 7
I summarizesrepresentative
data showingthe dependence
of /<oM
o n t y p i c a l b u f f e r s ( H O A c / O A c , H 2 P O 4 - / H P O 4 2, a n d
HCO3 lCOt') used to determine kHA. Rate constantswere
dcterminedas a functionof the concentrationof the acidic componentof the buffer. In the caseof phosphatebuffer, the slope
of a plot of kpapsobour. IH2PO4I yieldedksrpo,.:the intercept
a t I H 2 P O 4 - ]= 0 y i e l d e dt h e q u a n t i t y ( k s I H + ] * k s , 6 ) ( e q l ,
Figure l). Repeatingthis procedureat severalvaluesof pH and
= 0) vs. [H+]
plotting the resultingvaluesof /<pop11oM([H2PO4-]
gave a straight line with slopefts and interceptks,o at IH+] =
0 ( e q l . F i g u r e2 ) .
This procedurewas repeatedfor a number of different buffers
and Lewis acids,although not necessarilyusing as many points
4892 J. Am. Chem.Soc.,Vol. 103,No. 16. l98l
'Iable
I.
Wong and Whitesides
R a t c C o n s t a n t sf o r G e n c r a lA c i d - C a t a l y z c dl ) c c o r n p o s i t i o no f ' N A D ( P ) l J ( e r 1l - 3 ) o
T,,, ,c 11
HAb
PKa
H.o*
c H3c oc o ' t{
HCO,H
c H3c HoH c o ' H
cH3corH
1 . 74
2.5
3.8
3.9
4.8
5.4
6.1
6.2
6.2
6.5
'7.0
citric acid
G - 6 - P( R O P O , H - )
6 - P G( R O P O . H ' , )
maleicacid
P P i( H r P r O ? 2 - )
ImH*
McImH*
NaPi
g u a n i d i n i u r nP 1
I{epes
TRA
Tris
HCO3HrO
Pi/HOCHTCH,OH
Pi/glycerol
Pi/sorbitol
Pi/PEG
Pi/McrSO
Pi/DN,ttr
Pi/PA
Pi/tntoll)
t.l
"t.2
7.5
7.8
8.1
10.3
15.5
kg4. M-' lr-'
NADII
[ 9 . 4x 1 0 3 . f. r H +(l 7 . 3X 1 0 3 ) d
260
,r9)d
NADPH
32(ulAl = 0)
3l
31
690
460
340
320
210
180
s9 (60)n
59
r72
78
130(tBO)e
t40 (22Ue
27 Q9)e
(27)n
1 5 0( 1 7 0 ) e
2 8 0( 3 3 0 ) e
3 3 0( 3 4 0 ) €
5 2 0( 8 0 ) e ' r
690
(30)n
(30)o
(29)e
Q9)e
(38)n
(38)n
(31)o
(29)e
0.9)d
2 . s( 4 . q d
1 . 2( 2 . I ) d
0.97
0.96
0 . 2 2r c . 2 r d
0 . 3 3( 0 . s 4 ) d
0.087
0.068
0.4rrc.6rd
0.048
0 . 0 18
0 . 0 12
0.0081
x 1 0 - u= f t H , o l( 1 1 . 7X 1 0 - 6 )
11.26
a t
t l
30
28
2t (2t)"
22
28
24
27Q9)e
27 Q9)e
1 3( 1 3 ) e
(l5)€
28 (29)n
30 (32)e
3r (34)e
32 (28)e
32
Q2)e
(t4)n
(14)e
(12)e
( 14 ) n
(l6)o
(14)e
(13)o
(eq I)refcrto the ilisappearance
ofthe NAD(P)Hch.omophorc(0.I mM)at 340nm.
" ? - 25 "C for all measurefients.Rateconstants
C)rganiccosolventswere prescntat concentrltionsof 20%v/v (c\ccpt lbr sorbitol and polycthylencglycol, which werc 20%w/w). b Abbrcviltions: Hepes,
/V'2+ydrorycthylpiperazine_try''-2-cthanesulfbnale
i TRA. tricthanolamine:
Tis. tris(hydroxymethyl)aminome
thane;EG.
glycol;G-6-P,
cllrvle'ne
glucose
6-phosphatc;
6-PG,6-pho$phogtuconatc:
lm, imidazole:Mclm, methylimidazole;
glycol)
PDG,poly(ethylene
c
T
h
f
+
s
t
i
n
r
r
t
.
'
d
h
a
l
f
l
i
f
e
f
o
r
d
i
s
a
p
p
c
r
r
r
n
c
c
(
'
l
N
A
D
r
P
)
H
i
n
a
s
o
l
u
l
i
o
n
c
o
n
l
a
i
n
i
n
s
0
.
l
M
l-prupanol.
t l AL Hl .At ,l i l i . 0 .
ry']!90),PA
.
f 2 5 " C a V a l u e s o f r a t c r , \ n s t : r n t i i n p a r c n d r c s ( s r r c f r o m J , ' h n s r r n a n d T u la4z)o. na{ rl lcl lc l s u r evd! l u c ro f r , , . f T h cd i f f c r c n cbcc lweenobservcd
and calculated
valucsofr in thisinstanccis probablyduc to contributionsfrom catalysis
by HrCO..
as for H2PO4-. For each acid, measurementswere carried out
over a range in pH of approximately 2 units, bracketing its pKr.
Table I givesvaluesfor ksa obtainedfor thesebuffers and acids
and for solutionscontaining other components. The measured
rate constantsftHAwere usedto calculatethe half-life (r11) for
disappearance
of NAD(P)H in a solutioncontainingrhe acid HA
( p H 7 , t H A l + [ A - ] = 0 . 1 M , 2 5 o C ) ; f o r c o n v e n i e n c teh, e s e
valucsare also listed in Table I. In the instancesin which the
measurementsof ksa reproducethose reported by Johnsonand
Tauzon (shownin bracketsin Table I), agreementis adequate.
Figure 3 showsthe Bronstedplot derivedfrom thesedata (a =
0.54; lit.'a a = 0.47) and the relation betweenthe lifetime of
NAD(P)H in solutionand the pK" of the buffer acid. Theseplots
provide a check on the consistencyof the data and offer a way
of estimating the efficiency of other solution or buffer components
in catalyzingthe decompositionof NAD(P)H. ln particular,the
most effective catalyts for this decompositionwill be those for
which the pK" of the acid is approximatelynumericallyequalto
the pH of the solution(for H2POa, pH 7). This conditionreflects
a compromisebetweenacid strength (strongeracidsare intrinsicallymore effectivecatalysts)and degreeof ionization(stronger
acids havea greatertendencyto cxist as the catalyticallyinactive
conjugateanionsat a particular pH).
The practicalconsequence
of this analysisis the conclusionthat
acids having pK" values of -7 should provide the most rapid
catalysisof the decompositionof NADH. The most important
of theseacid catalystsfor the regenerationsystemdiscussedhere
are the phosphatederivatives(HOPO3H , ROPO3H ). The
catalytic efficiencyof thesespeciesis a disadvantagefor the system,
becauseG-6-P,6-PG, and P; (prescntas an impurity in the G-6-P)
are all necessarilypresent. All acceleratethe decompositionof
NAD(P)H.
We briefly examinedthe ability of organic cosolventsto stabilize
N AD ( P) H against phosphate-catalyzeddecompositionand found
only small effects (Table I). This observationis interestingin
light of the discoverythat attachmentof organicpolymerchains
'-l
{d
. z-,
\o
;-l
ov'
---_=j
HCO2H
,".il--.20"'
= o.i A
AcOH
T
t -2'
,/l\'
im
ot
Y]
n e' D
a \e* so .
)
a- \
Hcor
Tris
I
-6+
r l
IOOO1
+
I H.o
:
l
c
=
=
o
I
IOO;
B
cae$ i".
i
lo-j2A
P 'v r
HCO"H
HCo;
' . / ' d'i;t;
^$,,,.o
o.o"
oJ e\;Jl^ o
a ^^ lnF
Pi
I
r-TTT
?468tOt2
PKo
o
H^O
I
.r1.
A..e-
i
T
A]
'--l
t-
t4
i
----'1
t6
Figure 3. (A) Bronsted plot for acid-catalyzeddecompositionof NAD( P ) H . L e a s t - s q u a r ea
s n a l y s i so f t h e p o i n t s g e n e r a t e dt h e s o l i d l i n e ,
d e s c r i b e db y t h e e q u a t i o n l o g k s o = - 5 . 4 4 p K u + 4 . 0 9 . ( B ) P l o t s o f
calculatedhali-liic vs. pK" for decompositionof NADH (O) and NADP H ( A ) i n 0 . 1 M b u f f e r , p H 7 . 0 , 2 5 o C . T h e o b s e r v e dv a l u e sa r e i n d i c a t e db y ( O ) f o r N A D H a n d ( a ) f o r N A D P H . A b b r e v i a t i o n s : P y r ,
pyruvic acid; Lac. lactic acidl M, maleic acid; Melm, l-methylimidazole;
I m , i m i d a z o l e ;T R A . t r i e t h a n o l a m i n eT; r i s , t r i s ( h y d r o x y m e t h y l ) a m i n o methane; Hepes, N- 2-hydroxyethylpi perazine-N'-2-ethanesul
fonate.
to the adenine moiety of NADH provides significantly increased
stability. This stabilization apparently is not due to a general
solvent effect, since addition of high concentrations of organic
cosolvents does not have the same effect.
J. A m. C hem.S oc.,V ol . 103,N o. 16, 1981 4893
,\,fAD(P)H Cofactor Regeneration
t 1
, r-
I
o..
l -
d..
..
o-
o\..
.
t---
-j'-.o
3.
!;-o
o
\o
o
8
o
51-
12
-_-
1E
c r-t
on
Figure 4. Difference betweenthe observedrate constantsfor decompos i t i o no f N A D H ( 0 . 1 m M ) a n d N A D P H ( 0 . 1 m M ) a s a l u n c t i o no f p H
( 2 5 o C , 5 0 m M b u f f e r ; p H 3 . 8 5 . 8 , s o d i u m a c e t a t e ;p H 6 . 5 8 . 8 , T R A ;
pH )9, sodium bicarbonate). The solid line was calculated,by using eq
3 with PK.*ooo" = 6.2,ftl = 0.15h l.
F i g u r e 6 . R a t e c o n s t a n t st o r d e c o m p o s i t i o no f N A D ( r ) , N A D P ( t r ) ,
N A D H ( O ) . a n d N A D P H ( O ) ( 0 . 1 m M e a c h )a t 2 5 o C i n 5 0 m M
b u f f e r ; p H 3 . 8 - 5 . 8 ,a c e t a t c :p H 6 . 5 - 8 . 8 .T R A ; p H ) 9 , b i c a r b o n a t e .
iOO-r<-F.---rE=a=-o-
tTl
l
o.....--o--6
I
-l
T5
t
N
r.t
t\
l
,]
r
:
:
--*-
......-==-------{
E
E
s
048
r00
'-o
J
/CDH
-l
-l
I
t 0 2 [ t m H + ) u,
Figure5. Plotsof the ratesof decomposition
of NADH (O) andNADPt{ (O) against
theconcentration
of imidazolium
ion(lmlI+) at pH 7.0,
l-5 oC. The dottedlineswerethoseanticipated
if the magnitude
of koM
wasa linearfunctionof [mH*].
The decompositionof NADPH follows a different pattern from
that for NADH. Qualitatively,the factorsleadingto decompositionof NADH alsocontributeto the decomposition
of NADPH,
but there is in addition a large contribution to the rate that appears
to reflect intramolecular acid catalysisby the 2'-phosphategroup
of NADPH. The decompositionof NADPH is first order in
\IADPH (eq 2). The differencebetweenthe rate constantsfor
decompositionof NADPH and NADH is given by eq 3, where
Ku = 6.2 is the dissociationconstantof the 2'-phosphate,
and k1
= 0,15 h-t is a constantrepresentingintramolecularcatalysis.
Figure 4 showsthe data from which k1 was derived. At high pH,
wherethe 2'-phosphate
is entirelyionized,NADH and NADPH
d e c o m p o s ea t t h e s a m e r a t e ; a t l o w p H ( [ b u f f e r ] : 0 . 1 M )
NADPH decomposes
approximately3 times more rapidly than
NADH. The intramolecularinteractionof the 2'-phosphatewith
the dihydronicotinamidering proposedhere is consistantwith
p r e v i o u sN M R s t u d i e s . l T
Some buffers showeddeviationsfrom the Bronstedplot of
Figure 3 that were sufficientlylarge to be of practical interest
i n e f f o r t st o m a x i m i z et h e l i f e t i m eo f N A D P H i n s o l u t i o n : i n
particular, inorganicphosphateis more effectiveas a catalyst for
thc decomposition
of NAD(P)H than expectedfrom its pK" value,
and imidazole,Hepes,TRA, and Tris buffers were lesseffective.
lforeover, as observedpreviously,lathe ratesof decomposition
of NADH in theseorganicbufferswere not linear in the buffer
acid concentrationbut insteadfell off markedlyat high concentrationsof buffer (Figure 5). This observationmay be of some
practicalvaluein prolongingthe lifetimcoi NADPH. The origin
of this effect is not certain: it may reflect interactionsof the
organicbuffer with thc stackedconformationof NAD(P) Hr4 or
ilggregationof the buffer with a resultingdecreasein the rate of
protontransferto NAD(P)H, or someother effect. In anv event,
the /cp6valuesin Table I wereestimatedfrom the linearportions
( 1 7 ) O p p e n h e i m eNr . J . :A r n o l d ,L . . 1 .K a p l a nN
. . O.Bioc&emisrrt'1918
t7.2613-19.
o __t_--__
"lrl
0
T----Tr
roo
o
r00
200
T i m e(. h )
(o) andsoluble(o) enzvmes.GFigure7. Stabilities
of immobilized
, mL I gel,
6 - P D H :p H 7 . 6 ,5 m M M g ' * , 0 . 1M T R A ; i m m o b i l i z e4d U
s o l u b l e4, 5 U m L r s o l u t i o n .H L A D H : p H 7 . 0 .0 . 1 M p h o s p h a t e ;
9 U mL I solution.o-l-DH: pt{ 7.6,
immobilized,
2 U mL I gel,soluble.
0 . 1M T R A . 5 m M M g 2 +i:m m o b i l i z e dU. 4m L I g e l . s o l u b l eS.4U
M T R A , , 5m M M g 2 + : i m m o b i l i z e d ,
mL-rsolution
I C. D H : p H 7 . 6 , 0 . 1
werekept
2.2U mL I solution.Enzymesolutions
0.-5mL-r gel,soluble.
losses
in 5-mL vialsat roomtemperature
underair. The observed
in
inactivation.
boththermaldenaturation
andoxidative
activityrepresent
was avoidedby
the oxidativeinactivation
ln syntheticexperiments,
underargon.
keepingthe reactions
of plots such as thoseshown in Figure 5.
Stability of NicotinamideCofactors: NAD(P). The oxidized
nicotinamidecofactorsare more stablethan the reducedonesin
(SOrt , CN )'5 and/or reducing
thc absenceof strongnucleophiles
(measuredby
agentsfor pH <8-9 (Figure 6). Decompositions
enzymaticdeterminationof NAD(P)) follow first-orderkinetics
(eq a), with rate constantsthat are insensitiveto buffer compo-d ln
[NAD(P)] /dt = ftnoo,r)o*o
(4)
sition. The ratesof decomposition
are effectivelyindependentof
pH over the range 4-9. Similar resultshave been obtainedby
others.ls
EnzymeImmobilizationand EnzymeStability. Enzymeswere
immobilized by using proceduresdescribedelsewhereleby copolymerizationinto gels formed from poly(acrylamide-co-N( a c r y l o x y ) s u c c i n i m i d e( P
) AN-1000 or PAN-500) and trie t h y l e n e t e t r a a m i n(eT E T ) . I m m o b i l i z a t i o ny i e l d s w e r e s a t i s lactory (seeTable III). The stabilitiesof the immobilizedenzymes
were higher than thoseof the sameenzymesin solution(Figure
7). With the exceptionof G-6-PHD from I-. mesenteroides,20
a l l o f t h e e n z y m e su s e di n t h i s w o r k ( H L A D H , L D H , I C D H ,
( l 8 ) L o w r r .O . I L . P a s s o n n e aJu. V
. . : R t ^ - k ,M . K . J . . 1 .B i o l . C h e m .1 9 6 1 ,
) 3 6 . 2 3 5 69 .
; a x ,\ ' l ; B a u g h n R
, . : B l u m c n f e l dl -.{ . W
. . L.;Whitesides,G.
{ 1 9 ) P o l l a kA
\ 1 . J . j m . ( ' h e m . , S r ;1t ,9 8 0 l. 0 : . 6 3 2 4 - \ 6
( 2 0 ) I s a q u c .A . ; V i l h a r i s e n .\ ' 1 . : I e v r ' . I I . R B i o c h i m .B i o p h l ' s ,R e s .
( ' o m m u n .1 9 7 4 .5 e .q 9 4 9 0 1
4894 J. Am. Chem.Soc.,Vol. 103,No. 16. l98l
.OH^
-\-/v
\\
''HH-HE\ott
Hal
ArP*
l/lux)acx
Dt-==-aop--'-/
Ac
:-\-
-Acp
I
*ffi5o,
P6fii.7
'/,/,
,oH^
HO\,\
Ho- rc 6,
t
c-6-P
\P6l
pp
\r
,,\.-{--o. .oH
HCr
){
\_oH
Ho-
l *H
i Pase
I
-oH
("+-'-O\
,
'\
---t--\
uo-" riO j,a
^^
NT
--\--q '0H
XO'*o-.r-/tr-On
Dextrin , Storch
F-t.6-P2
Figure 8. Synthesesof glucose 6-phosphate(G-6-Pe. Abbreviarions:
HK, hexokinase;AcK, acetate kinase,PGI, glucosephosphateisomerase;
Pase,phosphorylasea; PGM, phosphoglucomutase.
Table ll.
( ' o n v c r s i o n t o ( i { - P w i t h P h o s p h o r y l a s ea n d
It,rtt,,r-t"."'rt
s t a rc h o
oyster gl1'cogcn
dertrin
p o t l t o s t a r c h .s o l u b l e
potato starch
c : o r ns t a r c h
rcl rc:activityb
G-6-P prod u c c d , cn r M
1.00
0.25
0.l8
36
26
0.r2
0 . 12
o S u b s t r a t2e' / ,( v ' l t t ' 1 i n0 . 2 M P i ( p H 7 . 2 1 . o R c s u l t s ' u v eorbct a i n c db y u s i n gt h cs o l u b l cc o l n p o n c not 1 ' t h cm i r t u r c . c T h c
L ' t l n c c n t r a t ioofn( l - 6 - Pu ' a sd c t c r r n i n cedn z y r n a t i c a lal yt ' t e r c a c tiLrncrf thc 2()',solution/srrs1'rcr.tsion
of substratc
in 0.2 NI phosphatc
s i t h i n r r r i o b i l i zP
1 l l - ) l b r 5 d a y 'w
c tal s c( 1 2 4L l )a n dP ( i M( 3 3 0L I ) .
'f
No udditional(l-6-Pproductit.rn
wasrtbscrvcd
al'tcr5 dey,s. he
r c c o vree dc n z ) 1 l naccrt i v i t i cw
s c r eP a s.e7 1 i tI P G M, 8 2 , i .
G-6-PDH, Pase,PGM, PGI, AcK) containthiol groupscloseto
or in the activesite and are sensitiveto autoxidation: reactions
were therefore conducted under argon.
Glucose6-Phosphate. Three routes to G-6-P were explored
(Figure 8). The selectivephosphorylationof glucosewith hexo k i n a s e( H K , E C 2 . 7. l . l ) a n d A T P , w i t h c o u p l e dr e g e n e r a r i o n
of ATP from ADP by usingacetatekinase(AcK, EC 2.7.2.1)and
acetyl phosphate.providesone practicalroute.e Thc cnzymesare
commerciallyavailableand inexpensive,
and acetylphosphatcis
easily prepared.6The glucose6-phosphatecan be isolatedanci
stored (as the barium salt), or used without isolation.
The enzymaticconversionof an appropriatestarchto glucose
(G- l -P) by phosphorylase
1-phosphate
(Pase,EC 2.4.1.l .)-catalyzed reactionwith phosphatcand subsequent
conversionof G- I -P
t o G - 6 - P w i t h p h o s p h o g l u c o m u t a(sPeG M , E C 2 . 7 . 5 . 1 )p r o v i d e
a secondprocedure, We have bricfly screenedpossiblepolysaccharidestarting materialsfor this reaction(Table II). The
forms of starch that seemto offer the bestcombinationof cost.
c o m m e r c i a la v a i l a b i l i t y ,a n d r e a c t i v i t ya r e p r e h e a t e d( 1 0 0 o C .
I min) solublestarchand dextrin. (Glycogenis the bestsubstrate
but is too expensiveto be usefulin most syntheticapplications.)
The equilibrium constantfor formation of G-6-P from G-1-P is
K = l 7 ( p I { 6 . 5 - 7. 2 , 3 9 o 6 ; ' z t t h u s , - 9 5 o /oof t h e e q u i l i b r i u m
m i x t u r e o f G - 6 - P a n d G - l - P c a n b e u t i l i z e db y t h e G - 6 - P D H .
Becausethe phosphorolysis
of starchrequiresa high conccntration
of phosphatein solution ([P1] >- 0.2-0.4 M), it is neccssaryto
isolatethe G-6-P producedin order to avoid unacceptablyrapid
phosphate-catalyzed
hydration of NAD(P)H. The prcsenceof
inorganicphosphateand unreactedstarch make the isolationless
( 2 1 ) C o l o w i c kB
. . P . ;S u t h e r l a n dE, . W . . / . B i o l . C h e m . 1 9 4 2 . 4 4 . 4 2 33 ' 7
Wong and Whitesides
convenientthan that usedin isolatingG-6-P preparedby hexophosphorylationof glucose.
kinase-catalyzed
The third route to G-6-P examinedbeginswith the conversion
(F-1,6-P2)to fructose6-phosphate
of fructose 1,6-diphosphate
(F-6-P)22with mild acid catalysis. F-6-P is convertedto G-6-P
(PGM): The equilibrium mixture
by using phosphoglucomutase
contains7W" G-6-P.22Isolationof G-6-P from the mixture is not
necessaryfor the purposeof NAD(P)H regeneration.The F-6-P
solutionis useddirectly in the presenceof PGI, G-6-PDH, and
the syntheticenzyme(seeExperimentalSection). This route is
preparations.sinceF-1,6-P2
not a practicalroute for large-scale
is moderatelyexpensive(-$80/kg). It may, however,be useful
in laboratorywork, sinceF-1,6-P2is commerciallyavailableand
no ATP regenerationsystemis required.
Two other routes leading to G-6-P were also exploredbriefly.
In one,direct reactionof glucosewith an anhydrouspolyphosphoric
acid2agave a mixture of glucosephosphates(including -14%
phosphorylationat the 6 position). Since G-6-P is resistantto
acid hydrolysis(reflux in 8.7 N HBr for l6 h resultsin only 1096
hydrolysis),other esterscan be destroyedwith little lossof G-6-P.
In practice,separationof G-6-P from phosphateand from other
impuritiesin thesemixturesprovedtoo inconvenientfor this route
to be practical. In the second,hydrolysisof starch to glucose-1-P
has been reported to occur in high yields by using an acidic
ion-exchange
resin(KUZ.EDEI0P).25 We werenot able to obtain
the specific resin used in this work, and our efforts with an
a v a i l a b l er e s i n ( D o w e x 5 0 ) l e d t o l o w c o n v e r s i o n(s- 1 0 % ) .
Of the severalproceduresexploredfor the preparationof G-6-P,
that basedon HK-catalyzedphosphorylationof glucoseby acetyl
phosphateseemsthe most convenient,primarily becauseisolation
of the product is simple. For most procedures,isolationis unnecessary,although it may be useful to reducethe volume of the
solutionby rotary evaporation(T - 35 'C). In certaincircumstances,however,isolationmay be required. The desirabilityof
isolationdependson the enzyme,on the method of preparation
of the G-6-P, and on the lifetime required for NAD(P)H (i.e.,
on the quantity of phosphatewhich can be tolerated). Acetate
ion is presentin G-6-P preparedby hexokinase-catalyzed
phosphorylation. Acetate is an inhibitor of horse liver alcohol dehydrogenase(HLADH, Kt = 9.5 mM)26 and must be removed
in reactionsinvolving this enzyme. This separationcan be effected
either by isolationof Ba(G-6-P) (seebelow) or by adjustingthe
G-6-P solutionto pH ry2 with H2SO4and concentratingat reduced pressureto volatilizeaceticacid. HCI cannot be usedin
this operationor other operationsthat would leave significant
concentrationsof chlorideion presentin solutionsto be usedwith
HLADH, sinceCl is alsoan inhibitor of this enzyme(K, = 100
mM for NAD and 30 mM for ketonesubstrates).27
Isolationof G-6-P as a solid is best accomplishedby preparing
the barium salt. The convenience
of this proceduredependsupon
the amount of phosphatepresentin the solutionbut is generally
straightforward. Although isolationsare describedin the ExperimentalSection,severalexplanatorycommentsare valuable.
Isolationsare typically accomplishedby addition of barium
chloride to precipitateexcessphosphateas barium phosphate,
separation.and addition of more barium chloride to precipitate
the barium salt of G-6-P. An alternativeprocedurefor removing
inorganicphosphateinvolvesits precipitationas magnesiumammonium phosphate.This procedureis also followedby isolation
( 2 2 ) P r e p a r a t i oonf F - 6 - Pb y h y d r o l y s iosf F - 1 . 6 - P w
2 ith I N HCl, 35 oC
for 5 days: Neuberg,C'.;Lustig,H.; Rathenberg,
M. A. Arch. Biochem.1943,
J. 33-44.
( 2 3 ) t - a r d y ,H . A . ; F i s c h e rH, . O . L . J . B i o l . C h e m . 1 9 4 61, 6 4 , 5 1 3 - 9 .
( 2 4 ) S c c g m i l l e rJ,. E . ; H o r e c k e rB
. . L . " / .B i o l . C h e m . l 9 5 l . 1 9 2 , 1 7 5 - 8 0 ;
V i s c o n t i n iM
, . ; O l i v e r ,C . H e l u . C h i m . A c t a 1 9 5 3 ,J 6 , 4 6 6 - 7 0 . Z u l e s k i .F .
R . ; M c G u i n n e s sE. , .T . J . L a h e l l e dC o m p d . 1 9 6 9 J. , 3 1 1 - 6 . F o r a m e t h o d
u s i n gP O C I 3 / ( M e O ) r P : O .s e e :S c h u t z n e rK, . ; C e s k .F a r m . 1 9 7 4 ,2 3 , 3 4 - 5 ;
( ' h e m .A b s t r .1 9 7 4 . 8 / . I 3 7 1 - 5 j .
( 2 - 5 )S i l o n o v aG. . V . ; L i s o u s k a y aN,. P . M e t o d v .S o t , r e mB. i o k h i m . 1 9 7 5 ,
6 7- 9 : C h e m .A b s t r . 1 9 7 6 .8 4 , I I 3 2 1L r .
( 2 6 ) S u n d .H . : T h e o r e l lt.i . I n " T h e E n z y m e s " , 2 n e
d d . ;B o y e r ,P . D . :
L a r d y 'F
, I . ;M _ " - r b a cK
h ., , E d s . ;A c a d e m i cP r e s s :N e w Y o r k . 1 9 6 3 V
; ol.7, pp
25 83
(27) ColemanP
. [ - . : W i e n e r .H . B i o c h e m i s t r t1, 9 7 1 .1 2 . 1 7 0 2 - 5 .
J. Am. Chem.Soc.,Vol. 103, No. 16, I98l
N AD( P)H Cofactor Regeneration
4895
I{eactions
TableIIL L:nzyrnatic
Useclto P r c p a r eD - L a c t i c rA c i d , T h r eo - 1 ) . ( + ) - l s o c i t r i cA c i d . a n d ( S ) - B e n z y l - a - t l ,A l c o h o l
D-lactic actd
r c a ct:o r c h a r a c t c r i s t i c s
s o l u t i o nv o l u m e ( L ) b
pl-I,tcnrp ("C)
r e a c t i o nt i n r e ( d a y s )
G.6-PDII
in'unobiliz-ationy icldc ( %)
i m m o b i l i z e du n i t s d
gelvolume(mL)o
r".,uu.ryf (%)
TNC
s y n t h e t i ce n z y n r e
i r n n t o b i l i z a t i o ny i c l d c ( % )
i r n r n o b i l i z c du n i t s d
gcl volumc (mL)"
r e c o v e r y t( % )
TNC
G - 6 - P( r n o l )
NAD(P) (mol)
TNC
r t ' n r a i n i n g /( %)
i s o l a t c dp r o d u c t s
g, purityh(%)
tttol
y'icld (7c) (bascd on)
ce (%)
6 - Pl t o s pl 1 1 l g cl ttt n ut t "
g, purity (71,)
tltoI
yit'ld front C;-6-P(ti)
(S)-bcnzy l-cv-d, alcoltol
i s o c i t r i ca c r d
3
7.8,25
4
2
1. s . 2 5
1
30
400
2.0
92
2 . 6x 1 0 7
I)LDH
40
300
2.0
94
4 . 3x 1 0 ?
0.60
5 x 10-4
l 200
B5
Zn (lac:tatc),
30
100
1.5
82
1 . 3x 1 0 7
ICDT{
56
100
25
86
8 . 0x l 0 s
0.12
2 x 10-4
l 020
50
B a .( i s o c i t r a t c ) "
5l
g5
0.42
7 0 ( p y r u v a t)c
95
I l a . (6 - P G ) .
2s0.92
048
u0
7 S ){
4
7u.8u
0.t1
s8 ( G - 6 - P )
I{n, (6-P(}).
96,84
0 . 23
15
30
400
2.0
90
2 . 5x 1 0 7
HLADH
60
400
13
86
1 . 0x 1 0 6
0 . 50
4 . 8x 1 0 - 4
10 0 0
16
PhCHD0ll
4 4 .8 0
0 . 38
80 (Ph(lDO)
95
lla. (6-PG),
2 1 5 9. 0
0.41
82
d R e a c t i o n sw c r e r u n w i t h p o s i t i v e p l l c o n t r o l u D a l c ra r g o n . D c t r i i s a r c g i v c n i n t h c c \ t \ ( r i r ) l c n t r l s r e t i o n . b T l r c s cv , , l u r n c s a r c t h o s c o t '
t h c s t i r r t i n gs o l u t i o n s . V o l u m c s a t t h c c ( ) n c l u s i o no f t h e r c a c t i o n w c r e - 2 1 7 l c r g e r d u c t o d d d i l i o n o l r c r g c n t s a n d r c i d o r b r s c t o n a i n t a i n
p l l - c I m n r o b i l i z a t i o n y i e l d = [ 0 0 ( o b s e r v e da c t i v i t y i n t h e e n z - y m e - c o n t a i n i nggc l ) / s t r r t i n g a c t i v i t y . d t i n i t
pnr('l min I. o Appro\imatc
v ( ) l u m eo f s w o l l c n e n z y D l c - c o n t a i n i n g c l u s c d i n t h c r c u c t o r . r " R c c o v c r y " r c p r c s r ' n t s t h c p c r . ( n t a g r o f t h ( ' c n T y m r t i c r c t i v i t l t h a t s u r v i v e s
t h c r e u c t i o n . " R c m a i n i n g " i s t h c p c r c c n t a g co f t h c N A D ( P X H ) p r c s c n t i n b i o c h c m i c i l l y a c t i v c l i r r m r t t h s c o n c l u s i o no f t h e r e r c l i o n . e T N
I t u r n o v c r n u m b e r ) = n l o l e s o t p r o d u c t l c n c r a t e d p e r m o l c o f c n z y m c ( c o l n c t o r ) o r i g i n L l l l yp r c s c n t , T h c l u f n o v c r n u n ] b c r s i l l e n o t c o r r c c t c d
i o r r c s i d u a lc n z y m c ( c o t i l c t o r ) a c l i v i t y f c m a i n i n g r t d l e c o n c l u s i o Do f t h c r c a c t i r n . h T h e q u l n t i t ! , o l l r o d u c t i s o l a t c d i s c i v e n b y " g " . T h e
T h u s . t h c l a c l i c r c i d p r c p a r a t i o ny i c l d e d
P c r c c n t a g co f d l i s m x t e r i t h a t c o u l d b c a s s i g n c dt h c i n d i c r t c d c o m p o r i t i o n i s g i v e n b \ ' " p u r i t \ " .
5 3 g o f s o l i d . t i n z y m . r t i ca s s a y si n d i c a t e d t h a t 5 0 . 4 g ( 9 5 2 ) o f l h i s c o u l d b c r s s i g n e dt h c s t r u c t u r c Z n ( D - l n c t r t c ) . . r n d 0 . 8 ! ( 1 . 5 7 ) Z n ( L l x c t r t c ) , . ! T h c q u r n t i t y o f s o l i d i s o l x t e di n g r a m s i s s i v c n b y " g " ; t h c l i r c t i o n o f t h i s m l r c r i r l l h l a s s i y \ r s t h c i n d i r a t c d p f ) d u c t i s g i v c n b y
''prrrity":
t h e c i r l c u l a t e dn u m b c r o f m o l c s o f l h i s p r o d u c t p f o d u c c d ( b a s c do n t h e a s s u m p t i o no f 1 0 0 1 7p L r r i t y )i s g j v e n b y " n l o l " .
of G-6-P as the barium salt. Barium saltscan inactivateenzymes
and precipitatewith some anionsand are toxic. They must be
removedbefore use of the G-6-P. Removal of barium is accomplishedby dissolvingthe Ba(G-6-P) in 0.1-O.2M H2SO4solution,
separatingthe insolubleprecipitateof barium sulfate,and neutralizing the resultingsolutionwith sodium hydroxide.
The stability of G-6-P, both under conditionsexperiencedduring
operationof an enzymaticreactorand during storage,is pertinent
to the use of this regenerationsystem. Figure 9 summarizes
of
semiquantitative
kinetic data dcscribingto the decomposition
seemedto follow first-order
G-6-P at 25 oC: thesedecompositions
kinetics. Figure 9 givesboth half-livesfor decompositions
and
first-order rate constants. On the scaleof times required for
completionof the syntheticreactions(-5 days),the decomposition
of G-6-P presentsno problem. For long-termstoragein solution,
decompositionis a modestproblem. By addition of ethyleneglycol
v f v). deor other organic componentsto thesesolutions(20o/o
compositioncan be slowedsignificantly: thesecomponentsare
often (but not always) innocuoustoward the enzymesused.28
Sorbitol,for reasonsthat are unclear,appcarsto dcstabilizeG-6-P.
Storage at 0 oC also greatly increasesstability (at 0 oC, the
hali-life for decompositionof 1 mM G-6-P in 0.1 M phosphate
buffer (pH 7) is 400 days).
Synthetic Applications of the NAD(P)H RegenerationSystem.
Wc havc tcstcd thc practicalityol thc NAD(P)H regcneration
systembascdon thc G-6-PDH from Leuconosticmesenteroides
i'or svnthetic applicationsby applying it in enzymc-catalyzed
preparationsof three materials: o-lacticacid,t hreo-o.(* )-isocritic
a c i d ,a n d ( S ) - b e n z y l - a - da1l c o h o l( F i g u r e 1 0 ) . o - L a c t i ca c i d i s
( 2 8 ) A s a k a u r aT
, ; A d a c h i .K . : S c h w a r t sE
. . J . B i o l . C h e n t .1 9 7 8 ,2 5 1 .
. . n c .1 9 7 9 .1 2 . 4 1 i 1 8 .
6 4 2 3 - 5 . S c h m i d ,R . D . A c d . B i o c h e m L
8
lo
oH6
first-orderrateconstants
half-lives
and observed
Figure9. Estimated
(kou'0,
Buffers:(o) 0.1 M
6-phosphate.
of glucose
for decompositron
(v) 0l M glycine.Organiccosolvents
(r) 0.1 M acetate,
phosphatc,
glycol;Gc,glycerol;
(20Vo
E, ethanol;
v lv) areasfollows:EG.ethylene
S, sorbitol.
DMSO,dimethylsulfoxide;
DM F.,V,rV-dimethylformamidel
(or upperlimits
lowerlimitsfor hali-lives
Pointswith arrowsrepresent
wasdetected
in thesesolutions
overthe
for kohd):no decomposition
courseof I month.
2
4
a useful synthon for the preparationof chiral substancesand
complements.t--Lacticacid is alreadyreadily availableby fermentation. Isocitricacid containsan array of chiral functionality
and may alsobe usefulas a startingmaterialin synthesis.Chiral
b e n z y l - d la l c o h o li s u s e d i n r e s e a r c hb i o c h e m i s t r y .T a b l e I I I
l e t a i l sa r e g i v e n i n t h e
s u m m a r i z e dr e p r e s e n t a t i vree a c t i o n s d
ExperimentalSection. A fervgeneralcommentson thesereactions
follow.
The enzymesin thesepreparationswere immobilizedseparately
a n d t h e e n z v m e - c o n t a i n i ngge l sm i x e d o n l y i n t h e r e a c t o r . T h i s
p r o c e d u r eg i v e ss l i g h t l yl.o w e ro v e r a l lr c a c t i o nr a t e st h a n o n r -i n
w h i c h t h e e n z v m e sa r e c o i m m o b i l i z e di n t h e s a m eg e l . l e l t r s .
Wong and Whitesides
4896 J. Am. Chem.Soc.,Vol. 103,No. 16, 1981
r, n \ : ^ -" " 2
H
T a b l e I V . V a l u c s o l K y ( n r M ) f o r N A D ( P ) ( F tl )o r
S c l e c t e dI r n z y n t c s
r ) n zy m c
.OH
\s'
o,cf|9%
,' -"2
.-
-q
ozcacoi
-
Hzo
I
Plo'
xo;p-;o-co;
Abbreviations:
basedon NAD(P)H regeneration.
Figure10. Syntheses
D-LDH, o-lacticdedehydrogenase;
G-6-PDH, glucose-6-phosphate
ICDH, isoHLADH, horseliver alcoholdehydrogenase;
hydrogenase;
citratedehydrogenase.
however,more convenientand permits independentcontrol of the
amount of each enzyme present in the reaction. We have not
explicitly determinedthe rate-limiting step in thesereactions. The
assayedactivities of immobilized G-6-PHD and the coupled
syntheticenzyme were approximatelythe same. We can only
estimatethe expressedactivity-that is, the activity under the
conditions of the synthetic reaction, rather than under the V^u
conditionsused in the assays-but we note that the quantitiesof
products isolatedover the indicated intervals in which the reactors
were operatingsuggestedaverageenzymaticactivities -30plo those
measuredunder assayconditions.
The solutionsused in thesereactionsare relativelydilute, and
the volumesrequired to generatea given quantity of product are
accordinglysomewhatlarger than thoseusually used in conventional organic synthesis.The use of dilute solutionsis dictated
by product inhibition: thus, K; = 0.13 M for lactate in the LLDH-catalyzed reduction of pyruvate30(Kr,,r= 0.019 M for
D-lactate in the D-LDH catalyzedreduction of pyruvate)3rand
Ki = 0.060 M for isocitratein the lCDH-catalyzed conversion
of a a-ketoglutarateto isocitrate.32
The workup of the reactionsconsistsof stoppingthe stirring,
allowing the gel particlesto settle(typically l-2 h), and decanting
the product-containingsolution. The gel can usually be usedagain
immediately. The acidic products in this work were usually
isolatedby conversionto metal saltsand precipitationwith organic
solvent. The product purities were typically 70-90olo;higher
purities required crystallization(zinc lactate, Ba3(6-PG)2)or
distillation (benzylalcohol). The initial conccntrationof NAD
or NADP in thesereactorswas -0.1 mM. This value is fixed
for NAD by the requirementthat it be aboveKy for G-6-PDH
(see Table IV). A lower concentrationof NADP could, in
principle,have been used. No effort was made to recoverthe
nicotinamidccofactorsat the conclusionof the reactions,although
their residualactivity was determinedby enzymaticassays.
Discussion
This methodfor regenerationof reducednicotinamidecofactors
relativeto other methods.
has both advantagesand disadvantages
Its advantagc,s
arc six: First, it can reduceboth NAD and NADP.
It is thus almostuniquelysuitablefor the few problemsthat might
involveboth typesof cofactorsimultaneously.rlMore importantly,
it providesa single convenientprocedurefor either cofactor
separately.Second,both G-6-P and 6-GP are innocuousto most
enzymes,unlike the reactivealdehydesor ketonesproduccdby
Third, the equilibrium
methodsbasedon alcoholdehydrogenases.
( 2 9 ) M o s b a c hK, . ; M a t t i a s s o nB, . A c t a C h i m .S c a n d . 1 9 7 0 , 2 4 , 2 0 9 3 - 1 0 0 ;
, assaC o s t a ,J . M . , P h . D .T h e s i s ,D e p a r t m c not f C h e m i c a lf l n g i n e r r i n gM
, A, 1980.
c h u s e t t sI n s t i t u t eo f T e c h n o l o g yC, a m b r i d g eM
( 3 0 ) S t a m b a u g hR, . ; P o s t ,D . J . B i o l . C h e m . 1 9 6 6 . 2 4 1 , 1 1 6t2.
( 3 1 ) D e n n i sD
, . ; K a p l a n .N . O . " / . B i o l . C h e m . 1 9 6 0l,J - t . 8 1 0 - 8
( 3 2 ) C l e l a n dW
. . E . i n " V e t h o d si n
, . W . : T h o m p s o nV, . W . : B a r d e nR
. . P . ; K a p l a n ,' r - . O . , E d s . :A c a d e m i cP r e s s :l . - e w
E n z y m o l o g y "C, o l o w i c k S
Y o r k , 1 9 6 9 ;V o l . 1 3 ,p p 3 0 - 3 3 .
NAD
0 . 1l
G-6-PDH(1,.
mcsentcroides)
formatc dchydrogcnase 0.09
(C. boidirtii)
a l c o h o ld c h y d r o g c n a s e 0 . 0 7 4
(yeast)
0 . 0 17
TILADH
1.24
D-LDTI
0.253
L-LDI-I
ICDH
g l u t a m a t cd e h y d r o g e n a s c 0 . 1
l i o o a n r i d cd c h v d r o s c n a s e 0 . 1 4
NADH
NADP NADPH TCf
0.0057
10
JJ
0.011
0.027
0.071
0.011
3+
35
35
0 . 0 0 0 1 0 . 0 0 1 J/,
0.025 36
37
0.024 0.047
5.0
o D c t e r m i n cidn t h i sl a b o r a t o r b
yy D. Abril.
constantsfor reducednicotinamide formation are high, because
the reaction is renderedessentiallyirreversibleby hydrolysis of
to 6-phosphothe initially formed 6-phosphogluconolactone
is comdehydrogenase
gluconate. Fourth, glucose-6-phosphate
mercially available,inexpensive,and easy to manipulate and
has
immobilize. Fifth, becauseG-6-PDH from I. mesenteroides
no essentialthiol groups,this enzymeis relativelystableunder
aerobic conditionsand can be used for regenerationof reduced
environments.Sixth,
nicotinamidecofactorsin oxygen-containing
the values of Ky for the oxidized nicotinamide cofactors for
systembased
G-6-PDH is not usuallyhigh, and the regeneration
on G-6-PDH thus requiresonly the concentrationof thesecofactors
that would be required for efficient operationof the synthetic
e n z y m e s( T a b l e I V ) .
This regenerationprocedurealso has severaldisadvantages.
First, althoughglucose6-phosphateis easilypreparedin quantity
from inexpensivestarting materials,it is intrinsically a more
complexsubstancethan the formate or simple alcoholsusedwith
other regenerationmethods. We note that this disadvantageis
not severein our laboratory, since the 6-PG producedis itself a
valuableintermediatefor the productionof pentosederivatives
( r i b o s e 5 - p h o s p h a t e r, i b u l o s e 5 - p h o s p h a t e r, i b u l o s e 1 , 5 - d i phosphate,NADl.ra'rs The ability of a particularlaboratoryto
use thesesubstancesdepends,however,on the researchbeing
conducted.Second,both G-6-P and 6-PG are generalacid catalysts for the hydration of the reducednicotinamidecofactors'
of G-6-P usedin this work, the lifetime of
At the concentrations
NADH is shortenedby alkyl hydrogenphosphatecatalyzeddecompositionto - l5oloof that in a phosphate-freesolution,while
that of NADPH is shortenedto approximately 70olothat in
solution. In the synthesisreportedhere,this dephosphate-free
creasehas not beenparticularlyimportant. The cofactorturnover
n u m b e r s( 1 0 0 0 - 1 2 0 0 )r e p o r t e di n T a b l e I I I w e r e c a l c u l a t e d
conservatively:that is, they are basedonly on the quantity of
NAD(P) originallyaddedto the solutionand containno correction
for the quantity of cofactor remaining at the end of the reaction.
In a reactiondesignedto optimize cofactor utilization, either the
cofactorswould be recoveredor the reactionwould be run in a
way that resultedin essentiallycompleteconsumptionof cofactor.
one can estimatefrom the data in
Under thesecircumstances.
Table III that theseturnover numberswould range from 8000
(for o-lactic acid) to 2000 (for isocitric acid). Such turnover
numbersare high enoughfor most synthesesof complex molecules.
of the NAD(P)H
For slowerreactions(in which decomposition
( 3 3 ) S c h u t t e .H . ; F l o s s d o r f ,S: a h m , H . ; K u l a , M . R . I u r . J . B i o c h e m .
1 9 7 6 . 5 2 .l 5 l 6 0 .
( 3 4 ) W r a t t e n ,C . C . ; C l e l a n d ,W . W . B i o c h e m i s t r l1' 9 6 3 ,2 , 9 3 5 - 4 1 .
; asser,
( 3 5 ) Z e w e .\ ' . : F r o m n ,H . J . J . B i o l . C h e m .1 9 6 2 ,2 3 7 . 1 6 6 8 - 7 5 G
F . ; D o u d o r o f f .M . : C o n t o p o u l o sR. . J . G e n .M i c r o b i o l .1 9 7 0 '6 2 ' 2 4 1 - 5 0 .
( 3 6 ) F a h i e nL, . A . ; W i g g e r t ,B . O . : C o h e nP
, . P . " / .B i o l . C h e m , 1 9 6 5:,4 0 ,
1 0 8 39 0 .
( 3 7 ) M a s s e l 'Y
. i o p h t ' . sA. c t a 1 9 6 0 ,3 7 , 3 1 4 - 2 2 .
. . BiochintB
s .. M . J . . 4 m .C h e n t .S o c .
( 1 8 ) W o n g .( . t l . : l \ ' l c ( ' u r n ,S . D . . W h i t e s r d c G
1 9 8 0 .l 0 : . 7 9 1 8 q
( 3 9 ) W a l t ,D . R : R i o s - M e r c a d i l V
l o .M . : A u g e . . f: W h i t e s i d eGs.. M . i .
4 n t . ( ' h e m . . S r r il.9. f i 0 . / / ) - . .r 8 ( ) 5 6
NAD( P)H Cofactor Regeneration
J. Am. Chem.Soc.,Vol. 103, No. 16, I98l
4897
prepared at -30 oC by passinganhydrous NH3 over cooled MeOH for
30 min. Addition of acetyl phosphateto NH3/MeOH was performed
a t - 6 0 o C . T h e t e m p e r a t u r ed u r i n g a n d a f t e r a d d i t i o n i n t h e N H r /
M e O H t o t h e r e a c t i o nm i x t u r e s h o u l d b e - 1 0 o C o r b e l o w t o p r e v e n t
a m i n o l y s i so f a c e t y l p h o s p h a t e .
Rates of Phosphorolysisof starches and Dextrin. The polysaccharide
substances(2%, w lw) were added to water containing 0.2 M phosphate
(pH 7 .2). Of thoseexamined,only glycogenand dextrin were completell
soluble. For those substrateswhich were only partially soluble. the
supernatantsolution over the suspensionwas used. "Heat-treated" starch
was preparedby bringing the starch suspensionin water to 100 oC with
stirring, letting stand for l-2 min, and then cooling and adding an equal
volume of 0.4 M phosphatebuffer, pH 7 .2. To thosesolutions(2.5 mL)
were addedEDTA (0.3 pmol), MgCl2 (30 pmol), PcM (3 U), c-6-pDH
( 1 0 U ) , P a s e( r a b b i t m u s c l e ,I U ) , a n d N A D P ( 0 . 6 p m o l ) . T h e r a t e o f
phosphorolysiswas measuredby following the absorbancechange at 340
nm.
Kinetics of hydrolysis of glucose6-phosphatewere carried out with I
mM solutionsof glucose6-phosphate.Aliquots (0.1 mL) were withdrawn
periodically from solutionsmaintained under specifiedconditions,diluted
to 3 mL with 0.1 M TRA buffer, pH 7.8, and assayedenzymaticallyfor
G-6-P.
Kineticsof Decompositionof NAD(P)(H). Solutionsof NAD(p)(H)
( 0 . 1 m M ) w e r e p r e p a r e di n l 0 m L o f 5 0 m M b u f f e r a n d k e p r a t 2 5 o C .
A l i q u o t s ( 3 m L ) w e r e w i t h d r a w n p e r i o d i c a l l y a, n d t h e c o n c e n t r a t i o no f
N A D ( P ) H w a s d e t e r m i n e ds p e c t r o p h o t o m e t r i c a l lavt 3 4 0 n m . O c c a sional points were checkedenzymatically with GluDH. Decomposition
in organic solvent/water (2u% v/v) mixtures was carried out with the
buffer concentrationmaintainedat 0.1 M. The pH valueswere adjusted
after addition of the organic solventto the aqueousbuffer at the chosen
p H . F o r N A D ( P ) d e t e r m i n a t i o n sl,- m L p o r t i o n sw e r e w i t h d r a w n a n d
a d d e d t o 2 m L o f 0 . i M T R A , p H 7 . g , c o n t a i n i n gG - 6 - p ( 2 m M ) .
G - 6 - P D H ( 2 U ) w a s a d d e da n d t h e a b s o r b a n c ec h a n g ea t 3 4 0 n m w a s
measured.
Immobilization of n-Lactate Dehydrogenase(o-LDH). The comm e r c i a l l y a v a i l a b l eu - L D H i n I m L o f 3 . 2 M ( N H 4 ) 2 S O 4( 5 m g , 1 7 2 5
U) was dissolvedin 20 mL of 0.1 M phosphatebuffer, pH 7.0, which had
been cooled to 0 oC and deoxygenatedwith a stream of argon. The
solution was concentratedby ultrafiltration under reducedpressureat 0
Experimental Section
"C by using an lmmersible-CX Molecular Separator Kit (Millipore
perkin-Elmer552spectro_ General.UV spectraweretakenwith a
C o r p . ) . W h e n t h e v o l u m e w a s r e d u c e dt o a b o u t I m L , a n o t h e r 2 0 - m L
photomer,equippedwith a constant-temperature
cell. NMR spectra
portion of cold phosphatesolutionwas added and the mixture was again
weremeasured
at 250 MHz for determinations
of enantiomeric
excesses
concentrated. Three repetitionsof this processover 8 h resulted in an
(Bruker270). water wasdoublydistilledin glass.The pH of reaction
enzymesolution in l mL of phosphatebuffer having 1380 U of activity
mixtureswascontrolledwith a westonModet7561pH controllercou( 8 0 % ) w h i c h w a s r e a d y f o r i m m o b i l i z a t i o n .T o 0 . 6 g o f P A N - 1 0 0 0 w a s
pledto a Monostatcassette
peristalticpump.
a d d e d 2 . 4 m L o f 0 . 2 M H e p e sb u f f e r c o n t a i n i n g8 . 5 m M p y r u v a t ea n d
Materials.Enzymeswereobtainedfrom Sigma. NAD, NADp, di1 . 0 m M N A D H w i t h v i g o r o u ss t i r r i n g . A f t e r I m i n , 5 0 p L o f 0 . 5 M
thiothretol(DTT), 1,3-dithiopropanol,
dextrinandstarches,
androutine
DTT solutionwas added,followedby 300 rrL of 0.5 M TET solutionand
biochemicals
were from Sigma. PAN was preparedas described.re
the enzyme-containing
solution. The mixture gelledwithin 3 min. It was
Argonwasusedasan inert atmosphere;
welding-grade
materialwasused
kept at room temperature for I h. The gel was ground into small parwithoutpurification.
t i c l e si n a m o r t a r a n d w a s h e dw i t h 5 0 m L o f 5 0 m M H e p e s b u f f e r , p H
Assays.Assaysfor the followinggroupof enzymes
andbiochemicals
7 . 5 , c o n t a i n i n g5 0 m M a m m o n i u m s u l f a t e . T h e g e l w a s s e p a r a t e db y
weretakendirectlyfrom Bergmeyer:{lactatedehydrogenase
(LDH)
c e n t r i f u g a t i o na n d w a s h e da g a i n w i t h t h e s a m e b u f f e r w i t h o u t a m m o and lactate(pp a80, 1464),G-6-PDHand G-6-p (pp 458,1238),iso_
nium sulfate. A 20-pL aliquotof the gel suspension
in 30 mL of TRA
citratedehydrogenase
(ICDH) andisocitrate
(pp 624,1570),glutamate
b u f f e r ( 0 . 1 M , p H 7 . 5 ) w a s d i l u t e d i n 3 m L o f 0 . 2 M h e p e sb u f f e r , p H
dehydrogenase
(GluDH) and2-ketoglutarate
(pp 650, l5i1), horseliver
7 . 5 , a n d t h e e n z y m ea c t i v i t y w a s m e a s u r e d( 5 5 2 U , 4 0 % y i e l d )
alcoholdehydrogenase
(HLADH) (p 429),acetatekinase(AcK) and
The (NHo)rSOa presentin the original enzymepreparationcould also
acetylphosphate
(pp a25, 1538),phosphorylase
a (pase)(p 505),
be removedby centrifuging the enzymesuspension(5 mg in I mL of 3.2
phosphoglucomutase
(PGM) (pp 798),phosphoglucoisomerase
(pGI) (pp
M ( N H 4 ) 2 S O 4 , 4o C , 1 5 0 0 0 9 f, o r 5 m i n ) . T h e p r e c i p i t a t ew a s d i s s o l v e d
501, 113),hexokinase
(HK) (p 473),fructose6-phosphate
(F-6-p,'p
i n I m L o f 0 . 3 M H e p e sb u f i e r . p H 7 . 5 . a n d i m m o b i l i z e da s d e s c r i b e d
i 238),fructose1,6-diphosphatc
( F-1,6-P
p
1
3t
4),
6-phosphogluconate
z,
above.
(6-PG,p Da8). NAD(P)H wasdetermined
by usingGIuDH1p 2054:
Immobilization of lsocitrate Dehydrogenase(ICDH). The commerciar
NAD(P) wasdetermined
by usingG-6-PDHfrom L. mesenteroides
with
c n z y m ef r o m p i g l i v e r s u s p e n d e di n 5 0 % g l y c e r o l( 1 0 0 m g / 1 0 m L , 2 0 0
the sameprocedureusedfor NADP (p 2050).
U ) w a s d i a l y z e d a t 0 o C a g a i n s t4 L o f 0 . 0 5 M T r i s / 0 . 1 M N a 2 S O a
Assaysfor the activitiesof immobilized
enzymes
wereessentially
the
solution (pH 7.3) for 4 h, using a membranetube with molecular cutoff
sameas described.re
Aliquots(20-100rrL) of immobilized
enzyme
of 6000-8000 (spectrumMedical Industries,Inc., Los Angeles),and then
suspended
in buffer weretakenand addedto a 3-mL plasticcuvette
c o n c e n t r a t e da 1 0 o c t o 5 m L b y u s i n g a m o l e c u l a r s e p a r a t o rk i t a s
containing3 mL of bufferandthe requiredsubstrates.
Tie cuvettewas
describedabove. The resultingsolutioncontained I 80 u (9096yierd) of
stoppered
with Parafilmandshakenfor 3-4 s to mix the suspension.
The
e n z y m e . T h e p r o c e d u r ef o r i m m o b i l i z a t i o nw a s t h e s a m e a s t h a t u s e d
absorption
wasreadat 340nm for 5 s. Thisprocess
wasrepeated
about
ior o-LDH. Buffer [20 mL,0.2 M Hepescontainingisocitrate(50 mM)
I 5 times. A linearresponse
wasobtainedfor the changeof absorbance
a n d N A D P + ( 1 m M ) l w a s a d d e dt o 5 g o f P A N - 1 0 0 0 , f o l l o w e db y D T T
with time.
( 0 . 5 m L ) , e n z y m es o l u t i o n ,a n d T E T ( 3 m L ) . T h e g e l w a s w a s h e dw i t h
Acetylphosphate
waspreparedas the diammoniumsaltvia reactron
5 0 m M T r i s / 0 . 1 M N a 2 S O a / 5 0m M ( N H 4 ) 2 S O 4( 4 0 0 m L ) a n d t h e n
of anhydrousH3PO.with aceticanhydride.sAnhydrousH3pOawas
w i t h T r i s / S O a 2 b u f f e r ( 4 0 0 m L ) . T h e t o t a l a c t i v i t y o b t a i n e du . a s 1 0 2
madeby theslowadditionof P2O5( l9l .5 g) to stirredg5%HfO4 (500
U (56% yield).
g) at -10 oc. The mixturewaskeptfrom moistureat roomtemperature
Immobilization of Glucose-6-phosphate
with stirringfor at leastI h beforeuse. saturatedNH./MebH *u*
Dehydrogenase(G-6-pDH),
Glutamate Dehydrogenase(GluDH), and Horse Liver Alcohol Dehydrogenase(HLADH). G-6-PHD from l.. mesenteroide.rwas rmmo(40)_Bergleyer,H. U. "Methods ol Enzymatic Analysis"; Academic
b i l i z e d a c c o r d i n gt o t h e s a m eg e n e r a lp r o c e d u r e : l 0 m g o f e n z y m ep e r
_
P r e s s :N e w Y o r k . 1 9 7 4 .
g o f P A N 1 0 0 0w a s u s e d . G - 6 - P [ ( 6 m M ) a n d N A D p ( 0 . 6 m M t w e r e
is more rapid relative to production of product) or for reactions
in which maximum turnover numbers for the NAD(P) is important for economic or other reasons,the G-6-p- and 6-pGcatalyzedhydrationsmay becomeinfluential in determining the
choice of a regenerationsystem. Third, the 6-pG produced as
the product of oxidation of G-6-P must be separatedfrom the
product of the reduction. Although the separationsreported here
are straightforward, they are more complex than thoserequired
with formate/formate dehydrogenaseor ethanol/arcohordehydrogenase.
Theseproblemsnotwithstanding,our practicalexperiencewith
this and other NAD(P)H regenerationsystemssuggeststhat it
is one of the most convenient (perhaps the most convenient)
systemsavailablefor use in laboratory-scalesynthesis(that is,
syntheseson scalesup to severalmoles). The low cost and ease
of manipulation of G-6-PDH compensatefor the inconvenience
of preparing G-6-P. Formate/formate dehydrogenaseis a very
attractiveand useful alternativefor NADH regeneration,but the
c,ommercialenzyme is presently 35 times more expensivethan
G-6-PDH (for equivalentactivities),more sensitivetoautoxidation
than G-6-PDH, and limited to the reductionof NAD to NADH.
Ethanol/alcoholdehydrogenase
is not a particularryattractive
system: both acrtaldehydeand ethanoldeactivateenzymesrapidly,
and the equilibrium constantsfor reduction of carbonyl groups
by
_regenerationproceduresbased on this system are not high.
Chemical methodss'afor NAD(P)H regenerationlack ihe
specificityrequired to achievehigh turnover numbers. If reduction
of NAD(P) to NAD(P)H is 95% selectivefor hvdridedonation
to the 4 positionof the nicotinamidering, the iesiduaractivity
of the cofactor after 64 turnovers would be (0.95)6a= 4%o.We
require 103 turnovers in most synthetic applications. Thus,
presentlyavailableproceduresinvolving direct chemical or electrochemicalreductionof NAD(p) to NAD(p)H are insufficiently
selectiveto be practical.
4898 J. Am. Chem.Soc.,Vol. 103, No. 16, tgBt
presentduring the immobilizationto protectthe enzymeactivesite. Ater
the gel had formed, it was washedwith 50 mM (NH4)2SO4solutionand
5 0 m M H e p e s b u f f e r ( 0 . 2 M , p H 7 . 5 ) . T h e i m m o b i l i z a t i o ny i e l d w a s
3006, GIuDH was immobilized similarly ( l0 mglg of pAN- 1000) in the
p r e s e n @o f k e t o g l u t a r a t e( l 5 m M ) , A D P ( 2 m M ) , N A D H ( 5 m M ) . a n d
NH4CI (20 mM): NH4CI was added together with the enzymesolution
to minimize the reactionof NHa+ with PAN. The immobilization yield
w a s 4 0 % . F o r H L A D H , l 0 m L o f 0 . 3 M H e p e sb u f f e r , p H 7 . 5 , c o n t a i n i n g N A D + ( 2 . 5 m M ) a n d e t h a n o l( 5 0 0 m M ) , w a s a d d e dt o 2 . 5 g o f
PAN- 1000 with stirring, followed by addition of enzymesolution(50 mg
o f e n z y m ei n 2 m L o f H e p e sb u f f e r ) a n d T E T ( 2 . 1 m L ) . D T T w a s n o t
necessaryin this case. other procedureswere the same as above. The
immobilization yield was 6096.
coimmobilization of Phosphorylase a (Pase) and phosphogrucomutase
( P G M ) . P a s e( E C 2 . 4 . 1 . 1 . , 3 9U
0/30 mg) andpcM (EC 2.7.5.1,830
U l2 mg) were immobilized accordingto the generalprocedureby using
P A N - 1 0 0 0 ( 4 . 1 2 g ) i n t h e p r e s e n c eo f g l y c o g e n( l m M ) , p ; ( 7 0 m M ) ,
G - l - P ( 2 m M ) , G - 6 - P ( 5 m M ) , g l u c o s e1 , 6 - d i p h o s p h a(t1e m M ) , T E T
(2.7 mL), and DTT (0.3 mL). Other details were the same as those of
the general procedure. The immobilized yields were as foilows: pase,
3l%; PGM,40q6.
Coimmobilization of o-LDH, G-6-PDH, and phosphoglucoisomerase
( P G M ) . o - L D H ( 1 0 0 0 U / 3 m g ) , G - 6 - P D H ( 1 3 8 0U / 2 m g ) , a n d p G I
( E C 5 . 3 . 1 . 9 ,1 0 0 0 U l 2 m g ) w e r e c o i m m o b i l i z e ds i m i l a r l y b y u s i n g4 g
o f P A N - 1 0 0 0 i n t h e p r e s e n c e o fH e p e sb u f f e r ( 0 . 3 M , p H 7 . 5 , l 6 m L l
DTT (0.2mL), TET (3 mL), G-6-P (6 mM), F-6-p (1.5 mM), pyruvare
(8.5mM), NADP (0.6 mM), and NADH (l mM). The immobilized
y i e l d sw e r e a s f o l l o w s : o - L D H , 4 0 % ;G - 6 - p D H , 3 8 % ;p G I , 4 l % .
Measurementsof Enzyme Stability. Enzymes,either soluble or immobilized, in specifiedbuffer solutionswere incubatedaerobicallyat 25
oc without stirring.
Aliquots (20 pL) were withdrawn periodicaily,and
the enzymatic activity was determined according to the method described
in the assay section.
Glucose 6-Phosphate. Hexokinase-Catalyzed phosphorylation of
Glucose. G-6-P was prepared by HK-catalyzed phosphorylation and the
ATP regenerationsystem.eA 3-L solutioncontainingglucose( 1.4 mol),
ATP (10 mmol), MgCl2 (98 mmol), EDTA (4.8 mmol), and 1,3-dimercapto2-propanol (18 mmol) was deoxygenatedand maintained under
argon. Immobilized HK (1200 u) and AcK (1200 u) were addedto this
s o l u t i o n . D i a m m o n i u m a c e t y l p h o s p h a t e( 1 . 4 m o l , 9 0 % p u r i t y ) w a s
added to the stirred reaction solution in l0 portions over 60 h, and the
solution was maintained at pH 7.4 by addition of 4 M KoH solution by
using a pH controller. The reaction was performed at 25 oC. After 3
d a y s , 1 . 2m o l o f G - 6 - P w a s f o r m e d ( 0 . 3 6 4 M i n 3 . 3 L , y i e l d 8 5 % ) . T h e
solution was separated from the polyacrylamide gels by decantation.
Barium chloride (0.2 mol) was addedto precipitateinorganicphosphate;
this material was removed by filtration. The resurting solution (cont a i n i n g 0 . 3 6 4 M G - 6 - P ) w a s u s e dd i r e c t l y i n t h e f o l l o w i n gs y n t h e s i so f
isocitrate, after dilution with water. The turnover number for ATp
during the synthesiswas 140, and the recoveredenzyme activities were
as follows: HK, 92%: AcK. 80%.
For the preparationof Ba(G-6-P).?tl2o, a slight excessof BaCl2 (0.37
m o l ) w a s a d d e dt o I L o f t h e a b o v eG - 6 - p s o l u t i o n( 0 . 3 6 4 M ) i o i l o w e d
b y s l o w a d d i t i o no f 4 0 0 m L o f 9 5 % e t h a n o l . T h e p r e c i p i t a t e ds o r i d( l g 7
g) contained96% Ba(G-6-P).7[l20 (0.33 mol). This salt could be stored
in the refrigerator (4 "c) without dccomposition in the absenceof
moisture.
F o r r e m o v a lo f b a r i u m , t h e b a r i u m s a l t ( 1 8 7 g ) w a s s u s p e n d c di n 1 . 7
L of 0.2 M H2so4 and stirredvigorouslyat room temperaturefor 30 min.
The precipitated barium sulfate was removed by filtration or centrifug a t i o n . T h e f r e e G - 6 - P a c i d s o l u t i o nw a s t h e n n e u t r a l i z e dw i t h s o d i u m
hydroxide. A solution prepared by this procedure was used in the
H L A D F I - c a L a l y z , e ds y n t h e s i so f ( S ) - b e n z y l - a - d ya l c o h o l ( s e e b e l o w ) .
For the preparationof a concentratedG-6-p solution,I t_ of the G-6-p
solution (0.364 M) was acidified to pH 2.0 by using conccntratedH2SO4
o r H C I a n d c o n c e n t r a t e du n d e r r e d u c e dp r e s s u r e( a s p i r a t o r ,2 0 m m H g )
a t 3 5 o c t o 2 0 0 m L . T h e c o n c e n t r a t i o on f G - 6 - P i n t h e r e s u r t i n gs o l u t i o n
w a s 1 . 8 2 M . N o d e c o m p o s i t i o no f G - 6 - P w a s o b s e r v e dd u r i n g t h c p r o cess,and most of the acetateproduccdlrom acetyl phosphateevaporated
d u r i n g c o n c e n t r a t i o n . T h e c o n c e n t r a t e ds o l u t i o nw a s s t a b l e i n t h e r e frigerator: No decompositionwas observedover l month. This material
w a s u s e d i n t h e o - l a c t a t ep r e p a r a t i o nd c s c r i b e db e l o w .
Glucose6-Phosphate. Phosphorolysisof Dextrin. To a l-L- sorution
c o n t a i n i n g0 , 4 M p h o s p h a t eb u f f e r , p H 7 . 0 , a n d 4 0 g o f d e x t r i n w e r c
a d d e d p h o s p h o r y l a sae ( P a s e ,2 4 0 L j ) a n d p h o s p h o g l u c o m u t a s( e
pGM,
6 0 0 U ) c o i m m o b i l i z e di n p o l y a c r y l a m i d eg e l sa n d 3 m m o l o f D T T . T h e
reactionmixture was kept under argon with stirring at 25 oC. Enzymatic
a s s a yi n d i c a t e dt h e p r e s e n c e
i n s o l u t i o no f 7 8 m m o l o i G - 6 - p a f t e r 5 d a _ v . . s
M g C l 2 ( 0 . 4 m o l ) w a s a d d e dt o t h e s o l u t i o n .. \ q u e o u sa m m o n i a( . 1 0m t _
o i l 4 % s o l u t i o n )w a s a d d e ds l o w l ' t o t h c c o o l c d .s t i r r e ds o l u t i o n . T h c
Wong and Whitesides
resultingprecipitateof MgNHoPOa was removedby filtration. Ethanol
(95%, 400 mL) was addedto the filtrate, and the precipitateof unreacted
dextrin was discarded. Barium chloride (0.1 mol) was added to the
s o l u t i o na n d s t i r r e d f o r 3 0 m i n a t 0 . 4 o C . T h e p r e c i p i t a t e ds o l i d ( 3 5 g )
w a s i s o l a t e d . T h i s m a t e r i a l c o n t a i n e d8 2 % o f B a ( G - 6 - P ) . 7 H 2 O( 2 9 %
,vieldbasedon the number of glucoseunit in the starting dextrin). The
recoveredenzyme activities were as follows: Pase, 80%; PGM, 86%.
Glucose 6-Phosphate: Phosphorolysis of Soluble Starch. A suspension
of 40 g of solublestarch in I L of water was stirred and heatedto boiling
(it took about 30 min to bring the solution to a boil). The opaque solution
was allowed to remain at 100 oC for l-2 min. The mixture was cooled
to room temperature. To the resulting solution was added 0.4 mol of
NaH2POo. It was adjustedto pH 7.0 with 5 N NaOH. The solutionwas
deoxygenatedand maintained under argon. DTT (3 mmol) and the
enzymes recovered from the dextrin phosphorolysisdescribed above
( P a s e ,1 9 0 U ; P G M , 4 9 0 U ) w e r e a d d e d t o t h e s o l u t i o n . T h e r e a c t i o n
was performed by using the same conditions described for dextrin
phosphorolysis.After 5 days, no further reaction was observed,and the
G - 6 - P ( 9 6 m m o l ) f o r m e d w a s i s o l a t e da s b a r i u m s a l t i n t h e s a m e w a y
as describedabove: 43 g of solid was obtained which contained 70 mmol
of Ba(G-6-P).7H2O (86% purity), correspondingto a 32% yield based
on the number of glucose units in the starch. The recoveredenzyme
activities were as follows: Pase,76%; PGM, 78%.
Preparation of Fructose 6-Phosphatefrom Fructose 1,6-Diphosphate.
F - 1 , 6 - P 2 C a(22 6 0 g , 0 . 4 7 m o l ; p r a c t i c a lg r a d e f r o m S i g m a , 7 5 % p u r i t y )
w a s d i s s o l v e di n 1 . 5 L o f 2 N H C l . T h e s o l u t i o nw a s k e p t a t 8 5 - 9 0 " C
( u s i n ga s t e a m b a t h o r w a t e r b a t h ) w i t h s t i r r i n g f o r 1 . 5 h . T h e b r o w n
solution was treated with 60 g of activated charcoal and filtered to remove colored materials. To the colorlesssolution were added 107 g of
p o t a s s i u mo x a l a t e ( 0 . 9 4 m o l ) a n d 2 0 0 g o f B a ( O H ) 2 ( 0 . 6 4 m o l ) . T h e
r e s u l t i n gs o l u t i o nw a s a d j u s t e dt o p H 6 b y t h e a d d i t i o n o f 5 N N a O H
with stirring, and the precipitatedcalcium oxalateand barium phosphate
w e r e r e m o v e db y f i l t r a t i o n . T h e f i l t r a t e ( 1 . 5 L ) c o n t a i n e d0 . 3 3 m o l o f
F - 6 - P ( 7 0 % y i e l d ) . T h i s s o l u t i o nw a s s t o r e di n t h e r e f r i g e r a t o r( 4 " C )
for use in subsequentreactions.
Determination of Optical Purity. Benzyl-a-dl alcohol and o-lactate
were convertedto their MTPA estersarby using freshly prepared (*)( 2 R ) - a - m e t h o x y - ( 2 - ( t r i ful o r o m e t h y l ) p h e n y l ) a c e t ycl h l o r i d e. Z i n c o lactate (0.5 g) was first convertedto its methyl ester by using anhydrous
m e t h a n o l( 1 8 m L ) a n d d r i e d D o w e x 5 0 W ( H + f o r m , I g ) a s c a t a l y s t .
Type 3,A molecular sieveswere added,and the mixture was refluxed for
4 h. After filtration, methanol was removed by evaporation and the
methyl lactate was converted to the MTPA ester. The enantiomeric
p u r i t y w a s d e t e r m i n e db y ' H N M R e x a m i n a t i o no f t h e p r o t o n so f t h e
methoxyl group at 250 MHz. The MTPA ester of the racemic alcohols
were used as referencesfor determining the shift values of each pair of
diastereotopicmethoxyl group.
The optical purity of n-lactate was also determined enzymatically.
The zinc o-lactate (90 mg) was dissolvedin l0 mL of glycine*hydrazine
b u f f e r ( 0 . 4 M , p H 9 . 0 ) . A 3 - m L a l i q u o t w a s t a k e n ,a n d r _ - l a c t a t e
dehydrogenase(10 U) and NAD+ (2 mM) were addedto the solution. The
increaseof absorbanceat 340 nm (read againsta referenceconsistingof
t h e s a m e s o l u t i o n .o m i t t i n g o n l y t h e e n z y m e ) w a s r e c o r d e du n t i l n o
c h a n g eo f a b s o r b a n c e( , - 5 h ) w a s o b s e r v e d . T h e c o n c e n t r a t i o no f L l a c t a t e i n t h e s a m p l ew a s d e t e r m i n e db y u s i n g 6 . 2 2 m M - t c m - r a s a b s o r b a n c ec o e f f i c i e n to f N A D H .
Isocitratewas determineddirectly by enzymatic methodswith ICDH
a n d N A D P . T h e i n c r e a s eo f a b s o r b a n c ea t 3 4 0 n m w a s m e a s u r e da n d
t h e c o n t e n to f i s o c i t r a t ew a s d e t e r m i n e d .
Synthesisof o-Lactate. A 5-L solution containing sodium pyruvate
( 0 . 1 2M ) , G - 6 - P ( 0 . 1 2M ) , M g C l 2 ( 5 m M ) , E D T A ( l m M ) , a n d 1 , 3 d i m e r c a p t o - 2 - p r o p a n o( 2
l . 5 m M ) w a s d e o x v g e n a t e dw i t h a r g o n . T h e
i m m o b i l i z e dc - 6 - P D H ( 4 0 0 U ) , D - L D H ( 3 0 0 U ) , a n d N A D + ( 0 . 5 m m o l )
w e r e a d d e d . T h c r e a c t i o nw a s c o n d u c t e du n d e r a r g o n a t 2 5 o C w i t h
s t i r r i n g a n d t h e p H m a i n t a i n e da t 7 . 8 b y a d d i t i o no f 2 N K O H s o l u t i o n
by using an automaticpH controllcr. After 4 days, the reactionwas
c o m p l e t e . E n z y m a t i ca s s a y si n d i c a t c dt h a t t h e s u m m e dc o n c e n t r a t i o n
o f N A D a n d N A D H w a s 8 5 % o f t h e o r i g i n a lv a l u e o f N A D . T h e
polyacrylamidegelswere allowedto settle.and the solutionwas decanted.
B a c l ' ( 0 . 9 , sm o l ) w a s a d d c d t o t h e d e c a n t e ds o l u t i o nw i t h s t i r r i n g . f o r lowed by I L of ethanol. The precipitatedsolid was collectedby filtration
a n d w a s h e dw i t h - 5 0 ee" t h a n o l , T h e p r e c i p i t a t e ds o l i d w a s c o l l e c t e db y
f i l t r a t i o n a n d w a s h e dw i t h 5 0 % e t h a n o l . A f t e r b e i n gd r i e d . t h e p r o d u c t
( 2 - 5 0g ) c o n t a i n e d9 2 o / o
t ' t [b a r i u m 6 - p h o s p h o g l u c o n a tceo. r r e s p o n d i n g
to
a uOvovield basedon G-6-P lddcd (seebelow). The filtrate and washings
i v c r e c o l l e c t c da n d c o n c e n l , r a t eudn d e r r e d u c e dp r e s s u r ea t 4 5 o C t o a
v o l u m eo f I I a n d t h e n a c r d i f i e dt o p F { 2 . - (r v i t hc o n c e n t r a t e H
d Cl. Some
( 4 l ) D a l e .J . , \ . . D u l l . D l ; M o s h e r .H . S . J O r g . C h e m .1 9 6 9 ,3 4 .
l54l 9
NAD(P)H
Cofactor
Regeneration
inorganic salt was removed by addition of 2L of ethanorand filtration
of the resulting precipitate. The filtrate was concentratedagain under
reduced pressureto a volume of 500 mL and neutralized to pH 6.0 by
addition of solid znco3. Ethanol was added to the hot (70 .c) solution
until turbidity appeared. After the solution was cooled,the precipitates
were removed by filtration and discarded. The filtrate was concentrated
to a volume of 300 mL and somezinc lactate precipitated. Ethanol (100
mL) was added to the suspension. After the suspensionwas cooled to
4 oc, the precipitatedzinc lactate was collectedby filtration and dried.
This material (53 g) contained 95% of zinc o-lactate and 1.5% of zinc
L-lactate by enzymatic assays. These values correspond to a givo enantiomeric excess and 7096 yield based on pyruvate. tH NMR spectra
(250-MHz) of the MPTA ester of o-lactate methyl estershoweda 95%
ee based on the signals of the methoxyl groups (3.660 ppm for the olactate ester and 3.560 ppm for the r--lactateester). The turnover number
for NAD in this experiment was 1200. The G-6-pDH and LDH activities in the residual gels were 92% and,9496,respectively,of the original
values.
Conversion of Barium 6-Phosphogluconate (6-pG) to 6-phosphogluconic Acid and Assay Procedure. Concentrated HCI (18 mL) was
added to a stirred suspensionof 0.2 mol of barium 6-phosphogluconate
(Ba3(6-PG)2,106 g, 90% purity) in H2O (l L). After the-solutionwas
stirred for l0 min, NarSO4Ql.2 g,0.22 mol) was addedto the clear
solution and the precipitated Basoa was removed by centrifugation or
filtration. An aliquot of the solution (5 pL) was withdra*n and added
to 3 mL of TRA buffer (0.1 M, pH 7.6) containing MgCl2 (5 mM),
NADP (0.4 mM), and 6-PGDH (5 U). The concentrationof 6-pG was
determined from the absorbancechange at 340 nm. This assay showed
0 , 1 9 m o l o f 6 - P G i n l . l L o f s o l u t i o n( 0 . 1 7 3 M ; y i e l d , 9 5 % ) .
For the preparation of NaCl-free 6-PG, the same quantity of Ba3(6PG)2 was mixed with 1.1 L of 0.2 M H2so4 and the mixture was stirred
for 30 min. The precipitatedBasoa was removedand the solution (1.2
L ) a s s a y e d : i t c o n t a i n e d0 . 1 8 8 m o l o f 6 - P C ( 0 . 1 5 7 M ; y i e l d , 9 5 % ) .
Benzaldehyde-a-dwas prepared from benzaldehyde-1,3-dithiane
by
exchangewith DrO and hydrolysis.a2 Dry hydrogen chloride was bubb l e d i n t o a s o l u t i o no f 1 , 3 - p r o p a n e d i t h i o( l 0 l m L , I m o l ) a n d b e n z a l d e h y d e( 1 0 2 m L , I m o l ) i n c h l o r o f o r m ( 7 5 0 m L ) a t 0 o C u n t i l t h e
mixture saturated (-5 min). After standing at 25 "C for 0.5 h, the
mixture was washedwith two 200-mL portions of water, three 200-mL
portions of l@6 KoH solution, and two 200-mL portions of water, dried
over anhydrousNa2Soa (50 g), and evaporatedunder reducedpressure.
The solid obtained was treated with l0 g of activated charioar and
crystallized from methanol (500 mL) to give 180 S ea@ of 2-phenyl1 , 3 - d i t h i a n em
, p 6 9 - 7 0 o C . T h i s m a t e r i a l w a s d i s s o l v e di n 1 . 6 L o f
anhydrous tetrahydrofuran (distilled over caH2) and cooled to -60-70
oC The system was
deoxygenatedwith argon. A solution of n-butyllithium in hexane(625 mL,l mol) was then introducedover a period of
I h into the dithiane solution by using a stainless-steel
cannula under a
slight positiveargon pressure. The temperatureof the reaction mixture
was maintained between*60 and -70 oc. After the solution stood at the
s a m e t e m p e r a t u r ef o r 6 h , D 2 O ( > 9 9 . 9 V od , 1 3 0 m L ) w a s a d d e ds l o w l y
with stirring and the solution was warmed to room temperature. The
s o l u t i o n w a s n e u t r a l i z e da, t 0 o c w i t h I N H C I ( - l
L) and tetrahydrofuran was removed on a rotary evaporator. The solution was ext r a c t e d w i t h t w o 8 0 0 - m L p o r t i o n so f C H r C l 2 / p e n t a n e( l : 1 , v / v ) . T h e
o r g a n i c p h a s ew a s w a s h e dw i t h I N N a H C O 3 ( 4 0 0 m L ) , w a . r e r( 4 0 0
mL), and saturated Nacl solution (400 mL). The residueafter evaporation of the solventwas recrystallizcdfrom methanolto give l4s g (0.gs
m o l ) o f 2 - p h e n y l - 1 , 3 - d i t h i a n2e--d , m p 6 9 - 7 0 o C . T h e d e u t e r i u m i n corporation was near 100%, based on the virtual absenceof the c-2
p r o t o n( 6 ( C D C l j ) 5 . 1 ) .
_A solutionof HgCl2 Q26 g, I .2 mol) in methanol/H2O (9:1, v/v) (600
mL) was added to a stirred warm mixture (-49 "C) of deuteriodithiane
p r e p a r e da b o v e( l l 9 g , 0 . 6 m o l ) a n d H g O ( i l 8 g , 0 . 5 4 m o l ) i n t h e s a m e
solvent mixture (3 L) in a 5-L flask. The mixture was refluxed with
stirring for 4 h under argon and then cooled to room temperature. The
white precipitatewas removedby filtration, and the filtrate was distilled
through a 40-cm Vigreux column until about I L remained. CH2CIzl
p e n t a n e( l : 1 , v / v ) ( l L ) w a s a d d e dt o t h e r e s i d u e ,a n d t h e s o l u t i o nw a s
washedtwice with 500-mL porrionsof 4 M NHooAc solutionand twice
with 500-mL portions of saturated NaCl solution and dried over anhydrous Na2Soa (80 g). solvent was removed by distillation as before
to furnish 300 mL of liquid, which was transferred to a 500-mL flask and
distilled again through a 20-cm Vigreux column under reducedpressure
\qZl Seebach,D.; Erickson,B. W.; Singh, G J. Org. Chem. 1966,31,
4303-4.
J. Am. Chem.Soc.,Vol. 103, No. 16, l98l
4899
(27 mmHg). The fractions of benzaldehyde-a-dwith boiling pint 77-79
o C w e r e c o l l e c t e d( 5 1 g , 8 0 % ) .
N M R a n a l y s i s( i n C D C I 3 ) s h o w e dn o
aldehyde proton absorption at 9.95 ppm.
Synthesisof (S)-Benzyl-a-d1 Alcohol. To a 3-L solution contained
0.5 mol of G-6-P (generated from the barium salt by treatment of
equivalentamount of 0.2 M H2SO4as describedin the G-6-P preparation), l0 mmol of MgSOa, 0.48 mmol of NAD, and 400 U each of
G-6-PDH and HLADH (theseactivitieswere basedon benzaldehydeas
substrate),benzaldehyde-a-d(0.48 mol, 51.4 g) was added dropwise over
2 days. The solution was kept under argon and the pH was controlled
at 7.5 by adding 4 N KOH using a pH controller. After 4 days, the
solution was decanted from the gel and extracted continuously with four
250-mL portion of ether. 6-PG was recoveredfrom the aqueousphase
as its barium salt in 82% yield as describedpreviously. The ether solution
was washed once with saturated aqueous NaCl solution, dried over
MgSOo, and concentrated under reduced pressure. The resulting liquid
was distilled through a l0-cm Vigreux column to obtain 44 g of benzyl-a-dyalcohol(8096),bp 110 'C (30 mmHg). The compoundwas pure
and the deuterium incorporation was almost 10096by tH NMR (60
M H z , C C l a ) : 6 2 . 1 ( s , I H ) , 4 . 6 ( t , I H , , I n c o = 1 . 8H z ) , 7 . 3 3 ( s , 5 H ) .
The optical purity of the (S)-benzyl-a-d, alcohol was determined by
250-MHz 1H NMR spectroscopywith the MTPA ester: the methoxy
group was monitored (3.501 ppm for (S)-benzyl-a-d1alcohol in CDCI3,
3.535ppm for (R)-benzyl-a-dlalcohol);ee= 95%. The turnovernumber
of NAD+ was 1000, and its residual activity was 8096. The reisolated
G-6-PDH and HLADH retained 9096and 86% of their original activities,
respectively.
Synthesisof threo-o(l )-Isocitrates. A 2-L solution containing G-6-p
( 0 . 1 5 M ) , k e t o g l u t a r a t e( 0 . 1 5 M ) , N H 4 H C O T ( 0 . 4 M ) , M g C l 2 ( 5 m M ) ,
MnCl2 (l mM), and 1,3-dimercapto-2-propanol
(3 mM) was kept under
CO2 and the pH was automatically controlled at 7.5. The immobilized
G-6-PDH (100 U), ICDH (100 U), and NADP (0.1 mM) were then
added, and the mixture was kept at the same pH under CO, with stirring.
After 4 days,the concentrationof isocitratewas 0.102 M (68% reacted)
and no further reaction occurred. The total concentration of NADP and
NADPH at the conclusionof the reactionwas 5096of the original value.
The gels were separatedby centrifugation. GIuDH (100 U), G-6-PDH
(100 U), and EDTA (a mM) were addedto the supernatentsolution,and
the solution was stirred under argon to consumethe remaining G-6-p and
ketoglutarate(-3 days at pH 8.0). The solution,after separationof gels,
was adjusted to pH 4.0 with concentratedHCI to remove bicarbonate
(liberatedas CO2), which would precipitatewith Mn2+ and influencethe
following isolationstep. Manganese(ll) dichloride(63 g, 500 mmol) was
added to the solution and the pH adjustedto 7.5 by adding 5 N NaOH.
Ethanol (300 mL) was added to precipitate 6-PG as irs manganese(Il)
salt (96 g). This solid contained84% 6-PG (correspondingto an overall
y i e l d o f 7 5 % ) . B a r i u m c h l o r i d e( 7 8 . 2 g , 3 2 0 m m o l ) w a s t h e n a d d e d t o
the mother liquid to precipitateisocitrateas the barium salt (78 g): this
material contained88% of isocitrateby enzymatic assay,corresponding
to an overall yield of 58%. The turnover number for the synthesisof
i s o c i t r a t ew a s 1 0 2 0a n d 1 5 0 0f o r t h e t o t a l r e a c t i o n . T h e r e s i d u a l a c t i v ities of the recoveredimmobilized enzymeswere as follows: G-6-PDH,
8 2 % ; I C D H , 8 6 % ; G - 6 - P D H ( s e c o n da d d i t i o n ) , 9 2 % ; G l u D H , 9 l % .
These three enzymescould be used repeatedlyor stored in the refrigerator
at 4 "C under argon.
Synthesisof n-l,actate from Fructose GPhosphate. Sodium pyruvate
( 3 6 g , 0 . 3 3 m o l ) , M g C l 2 . 6 H 2 A0 . 2 9 , 6 m m o l ) , a n d N A D ( 1 5 2 m g , 0 . 2
mmol) were dissolvedin the F-6-P solution (0.33 mol in L5 L) prepared
a b o v e . T h e s o l u t i o nw a s a d j u s t e dt o p H 7 . 8 w i t h 5 N N a O H a n d d e o x y g e n a t e dw i t h a r g o n . D T T ( 0 . 9 2 g , 6 m m o l ) a n d e n z y m e sc o i m m o b i l i z e di n 4 g o f P A N - 1 0 0 0 ( o - L D H , 4 0 0 U ; P G I , 4 1 0 U ; G - 6 - p D H . 5 2 0
U) were addedto the solution. The total volumewas 1.6 L. The reactlon
w a s c a r r i e d o u t i n t h e s a m e w a y a s d e s c r i b e df o r t h e G - 6 - P / G - 6 - P D H
system for n-lactate synthesis. After 2 days, the reaction was complete
a n d z i n c l a c t a t e w a s i s o l a t e d( 3 0 g ) i n 6 7 % y i e l d w i t h 9 2 % o f p u r i t y '
( d e t e r m i n e de n z y m a t i c a l l y ) . B a r i u m 6 - p h o s p h o g l u c o n a twea s i s o l a t e d
( l 3 l g ) i n 7 5 V oy i e l d w i t h 9 0 % p u r i t y . T h e t u r n o v e r n u m b e r o f N A D
was 1650and the recoveredenzymeactivitieswere as follows: pGI, 92%:
G-6-PDH, 90%;o-LDH, 90%.
Acknowledgment. We thank our colleagues A. Pollak for his
advice concerning immobilization, S. Moore for 250-MHz NMR
measurements, M. Patterson for advice concerning optical purity
determinations, and L. Perez for assistance in PAN preparations.
Professor Bryan Jones (Toronto) first pointed out to us the potential advantages of the L. mesenteroides G-6-PDH as a catalyst
for cofactor regeneration.