Louisiana State University
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LSU Historical Dissertations and Theses
Graduate School
1994
Reaction-Intermediate Analogues: Design,
Syntheses, and Inhibitory Studies With Carnitine
Acetyltransferase.
Guobin Sun
Louisiana State University and Agricultural & Mechanical College
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Sun, Guobin, "Reaction-Intermediate Analogues: Design, Syntheses, and Inhibitory Studies With Carnitine Acetyltransferase."
(1994). LSU Historical Dissertations and Theses. 5832.
http://digitalcommons.lsu.edu/gradschool_disstheses/5832
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R e a c tio n -in te rm e d ia te an alo g u es: D esign, sy n th eses, a n d
in h ib ito ry stu d ies w ith c a rn itin e a c e ty ltra n sfe ra se
Sun, Guobin, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1994
UMI
3 0 0 N . Z eeb Rd.
Ann Arbor, MI 48106
REACTION-INTERMEDIATE ANALOGUES:
DESIGN, SYNTHESES, AND INHIBITORY STUDIES
WITH CARNITINE ACETYLTRANSFERASE
A D issertation
S ubm itted to th e G raduate Faculty of the
L ouisiana S tate U niversity and
A gricultural and M echanical College
in p a rtia l fulfillm ent of th e
requirem ents for the degree of
Doctor of Philosophy
in
The D epartm ent of C hem istry
by
Guobin Sun
B.S., Peking U niversity, Beijing, P. R. China, 1983
M.S., Peking U niversity, Beijing, P. R. China, 1986
A ugust, 1994
To my paren ts, brothers, and sister for th e ir encouragem ent.
To m y wife, Xia Jin , for h er love, patience, and support.
ACKNOWLEDGMENTS
I would like to express m y g ratitu d e to Professor R ichard D. G andour
for his guidance, support, an d encouragem ent throughout th is work. I would
also like to th a n k Professor Robert P. H am m er for his kindness and
readiness to a ssist me after Professor G andour left LSU. The m em bers of my
g rad u ate advisory committee, Professors M ary D. Barkley, M arc A. Cohn,
N ikolaus H. Fischer, A ndrew W. M averick, an d E dw ard Zganjar have always
been helpful an d concerned.
I would like to th a n k Professor W illiam A. Pryor for providing me w ith
his laboratory an d facilities. I w ish to th a n k Mr. M arcus N aum an and Dr.
D avid V argas for th e ir assistance on NMR spectroscopy; Dr. F ra n k R.
Fronczek for solving th e X-ray crystal structures; Dr. Kevin L. E vans for
proofreading th is docum ent; and Dr. Rafael Cueto for his help w ith facilities
in Professor Pryor's laboratory.
A special acknow ledgm ent goes to Drs.
Noirfn Nic a' B h aird and Rona R. R am say of UCSF for th e ir collaborative
effort in assaying th e compounds we synthesized.
Finally, I would like to th a n k th e facility, staff, an d grad u ate students
in th e D ep artm en t of C hem istry for providing a n enjoyable working
envirom ent, leaving me w ith m any m em ories of m y life as a g rad u ate stu d en t
in B aton Rouge.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S........................................................................................ iii
L IS T O F T A B L E S .................................................................................................. vii
L IS T O F F I G U R E S ...............................................................................................viii
L IS T O F A B B R E V IA T IO N S ..............................................................................ix
A B S T R A C T .................................................................................................................x
C h a p t e r I.
i n t r o d u c t i o n ....................................................................1
1.1. G EN ER A L................................................................................................ 1
1.2. CARNITINE ACETYLTRANSFERASE..........................................4
1.2.1. CATs from different sp ecies................................................ 6
1.2.2. Amino acid sequences of CATs............................................ 8
1.2.3. Inhibition of C A T s................................................................. 9
1.2.3.1. CoA an alo g u es......................................................... 9
1.2 .3.2 . C arnitine an alo g u es............................................... 11
1.2.3.3. Bile acids....................................................................14
1 .2 .3.4. Conform ationally constrained
reaction-interm ediate an alo g u es........................ 15
1 .2 .3.5. O ther in h ib ito rs....................................................... 17
1.2.4. S u m m ary.................................................................................. 18
1.3. DESIGN OF INHIBITORS OF CARNITINE
ACYLTRANSFERASE......................................................................... 19
1.3.1. Considerations in the enzyme inhibitor d e s ig n ............. 19
1.3.2. Design of reaction-interm ediate analogues..................... 22
1.4. PRO JEC T GOAL................................................................................... 23
1.5. SPECIFIC G O A L .................................................................................. 25
C h a p te r
II.
s y n th e s e s o f in h ib ito rs
O F C A R N IT IN E A C E T Y L T R A N SF E R A SE ............... 26
11.1. RETROSYNTHETIC ANALYSIS...................................................... 26
11.1.1. T arget molecule I I .................................................................. 27
11.1.2. T arget molecule I I I ................................................................ 28
II. 1.3. S u m m ary ............................................................................... 31
11.2 . RESULTS AND D ISC U SSIO N ........................................................ 32
II. 2 .1 . Syntheses of (2R,6R)-, (2S,6S )-, (2R ,6S )-, and
(.2S',&R)-6-carboxylatomethyl-2-hydroxymethyl-
iv
2,4,4-trim ethylm orpholinium , I I ..........................................33
11.2 .2 . Syntheses of m ethyl (2S,6S;2R,6R )- and
(‘2 S, 6R;2R, 6S )-2-[6-(2-cyanoethyl )-4,6dim ethylm orpholinyl]acetate, 8 ........................................... 44
11.2.2.1. P rep aratio n of
(RS)-5-(N, ALdimethylamino )-4hydroxy-4-m ethylpentanenitrile, (R S )-10.......... 44
11.2 .2 .2 . Optical resolution of
(RS)-5-(A,N-dimethylamino )-4hydroxy-4-m ethylpentanenitrile, (f?S)-10.......... 45
11.2.2.3. P rep aratio n of (R)- and (S)-5-(N,Ndim ethylamino)-4-hydroxy-4m ethylpentanenitrile, (R)- and (S)-10.................47
11.2.2.4. A ttem pted preparation of (R)- and (S )3-(l-m ethyloxiranyl)propanenitrile,
(R)- and ( S ) - l l ...........................................................48
11.2.2.5. S tru ctu ral analysis of
CR)-2-hydroxy-3-iodo-2-methylpropyl
4-nitrobenzenesulfonate, (R)-2 3 ........................... 50
11.2.2.6. Syntheses of m ethyl (2S,6S;2R,6R )an d (2S, 6R;2R, 6S )-2-[6-( 2-cyanoethyl )4,6-dim ethylm orpholinyl]acetate, 8 .................... 52
11.2.3. S u m m ary....................................................................................56
II.3. EX PERIM ENTAL...................................................................................57
11.3.1. G eneral procedures..................................................................57
11.3.2. Procedures..................................................................................59
C h a pt e r III.
in h ib it io n
of
cat by fo ur
STEREOISOM ERS OF
6-CARBOXYLATOMETHYL2-HYDROXYMETHYL-2,4,4TRIM ETHYLM ORPHOLINIUM ........................................79
III. l.PREVIOUS W O RK............................................................................... 79
111.2.RESULTS AND D ISCU SSIO N .........................................................82
111.3.CONCLUSIO N ........................................................................................ 8 6
C h a p t e r IV . c o n c l u s i o n ...........................................................................87
IV. 1.R E SU LT S................................................................................................. 87
IV.2.FUTURE D IR E C T IO N ........................................................................ 8 8
BIBLIO G RAPH Y.......................................................................................................92
v
A p p e n d ix A.
c r y s t a l l o g r a p h ic
data fo r
(2S,6ft)-2-HYDROXYMETHYL-6(METHOXYCARBONYL)METHYL-2,4,4TRIMETHYLMORPHOLINIUM IO D ID E .....................103
A p p e n d ix B .
c r y s t a l l o g r a p h ic
data fo r
(2/?,67?)-2-IIYDROXYMETHYL-6(METHOXYCARBONYL)METHYL-2,4,4TRIMETHYLMORPHOLINIUM IODIDE
A p p e n d i x C.
c r y s t a l l o g r a p h ic
107
data fo r
(2R, (iS) -0 -C ARB OXYLATOME TH YL -2 IIYDROXYMETHYL-2,4,4TRIM ETHYLM ORPHOLINIUM ........................................110
A p p e n d ix D .
c r y s t a l l o g r a p h ic
data fo r
(2 R, 6 R )-6- CARB OXYLATOMETH YL-2 HYDROXYMETHYL-2,4,4TRIM ETHYLM ORPHOLINIUM ........................................113
Ap p e n d ix E.
c r y s t a l l o g r a p h ic
data fo r
METHYL (2S,GS;2R,GR)-2-[6-(2-CYANOETHYL)4,6-DIMETHYLMORPHOLINYL] ACETATE
116
VITA................................................................................................................................119
vi
LIST OF TABLES
Table 1.1.
CATs purified from different species...............................................7
Table 1.2.
V alues of K m of CATs for different s u b stra te s .............................. 8
Table 1.3.
CoA an alo g u es.......................................................................................10
Table 1.4.
Inhibition of CATs by etomoxiryl-CoA............................................ 1 1
Table 1.5.
Inhibition of CAT by carnitine an alo g u es...................................... 12
Table 1.6.
Inhibition of CAT by rigid carnitine an alo g u es............................13
Table 1.7.
Bile acids as inhibitors of C A T ......................................................... 14
Table 1.8.
Conform ationally constrained
reaction-interm ediate an alo g u es..................................................... 16
Table 1.9.
Inhibition constants of hem iacylcarnitinium s w ith CA T
17
Table III.l. Inhibition of 6-carboxylatomethyl-2-hydroxymethyl2,4,4-trim ethyhnorpholinium on CAT............................................83
vii
LIST OF FIGURES
Figure 1.1.
S tru ctu ral relationship betw een th e proposed tetrah ed ralin term ediate in th e acetyl tra n sfer reaction and the
stru ctu re of H A C ..................................................................................17
Figure 1.2.
Proposed tetrah ed ral-interm ediate and analogue in h ib ito r.... 2 2
Figure II. 1.
T ransition stru ctu res for ring closure reaction of 14.................38
Figure II. 2.
ORTEP draw ing of (2S,&R)-2-hydroxymethyl-6m ethoxycarbonylm ethyl-2,4,4-trim ethylm orpholinium
iodide.......................................................................................................40
Figure II.3.
ORTEP draw ing of (2R,dR)-2-hydroxymethyl-6m ethoxycarbonylm ethyl-2,4,4-trim ethylm orpholinium
iodide.......................................................................................................41
Figure II.4.
ORTEP draw ing of (2I?,dS)-6-carboxylatomethyl-2hydroxym ethyl-2,4,4-trim ethylm orpholinium ............................. 42
Figure II.5.
ORTEP draw ing of (2R,6R )-6-carboxylatomethyl-2hydroxym ethyl-2,4,4-trim ethylm orpholinium ............................. 43
Figure II. 6 .
ORTEP draw ing of (i?)-2-hydroxy-3-iodo-2-methylpropyl
4-nitrobenzenesulfonate
51
Figure II.7.
T ransition stru ctu res for ring closure reaction of 10.................54
Figure II. 8 .
ORTEP draw ing of (21S,,6S;2i?)6R)-2-[6-(2-cyanoethyl)-4,6dim ethylm orpholinyl] ac e ta te ............................................................55
Figure III. 1. Mode of su b strate recognition in CAT............................................ 80
Figure III.2. T etrahedral-interm ediate analogues and
th e ir K\ values w ith respect to (R l-carnitine.................................81
LIST OF ABBREVIATIONS
[a]
specific ro tatio n
calcd
calculated
CAT
carnitine acetyltransferase
CoA
co enzyme A
concd
concentrated
COT
carnitine octanoyltransferase
CPT
carnitine palm itoyltransferase
DBU
l,8-diazabicyclo[5.4.0]undec-7-ene
DMSO
dim ethyl sulfoxide
DNB
3,5-dinitrobenzoyl
ee
enantiom eric excess
FAB
fast atom bom bardm ent (in m ass spectrom etry)
m-CPBA
m-chloroperoxybenzoic acid
MMPP
m onoperoxyphthalic acid, m agnesium sa lt hexahydrate
Ms
m ethanesulfonyl (mesyl)
MTPA-C1
(R )-(+)-a-m ethoxy-a-(trifluorom ethyl)phenylacetyl chloride
PNB
4-nitrobenzoyl
satd
satu rated
THF
tetrah y d ro fu ran
ABSTRACT
C arnitine acetyltransferase (CAT) catalyzes the reversible tra n sfer of
short chain (less th a n six carbons in length) acyl groups betw een carnitine
and coenzyme A (CoA). This reaction likely m odulates the reserves of free
CoA and acetyl-CoA in each organelle and m em brane in ways specific to the
local dem ands. To probe th e stru ctures of the m olecular interactions between
carnitine and th e active site in CAT, we have designed and synthesized
conform ationally constrained reaction-interm ediate analogues, which not
only in h ib it enzym atic activity, b u t also help reveal the topography of the
active site.
The
syntheses
of (2R,6R )-,
(2S ,6 S )-,
(2R,6S)-,
and
(2S,6R)-6 -
carboxylatom ethyl-2-hydroxym ethyl-2,4,4-trim ethylm orpholinium
described.
are
The single-crystal X-ray structures of these compounds are
presented. These four stereoisom ers are tested as specific inhibitors of CAT.
The resu lts confirm th e hypothesis th a t there is two-point recognition by CAT
for carnitine and acetyl-CoA is the th ird locus for chiral recognition.
The
resu lts also strongly support th e proposed m echanism for acetyl tran sfer
betw een carnitine an d CoA.
The syntheses of m ethyl (2S,6S;2R,6R )- and (2S,6R;2R,6S)- 2-[6-(2cyanoethyl)-4,6-dim ethylm orpholinyl]acetate and the resolution of 5-(N,Ndim ethylam ino)-4-hydroxy-4-m ethylpentanenitrile are also described.
x
Cha pter
I
INTRODUCTION
1.1.
GENERAL
C arnitine (3-hydroxy-4-trim ethylam m oniobutanoate, see below), a
n a tu ra l constituent of higher organism s, is absolutely necessary for efficient
CU
p u
(C H l)3 K
2- C O RO
H
R = H; carnitine.
R = acyl; acylcarnitine.
m etabolism of long-chain fatty acids . 1 " 5 F atty acids are activated by forming
acyl-coenzyme A (acyl-CoA) on th e outer m itochondrial m em brane, w hereas
they are oxidized in th e m itochondrial m atrix.
Long-chain acyl-CoA
molecules cannot p en etrate th e in n er m itochondrial m em brane leading into
th e m atrix.
Acyl groups m u st be tran sferred to carnitine to form
acylcarnitine, which can p en etrate the m em brane. Once inside the m atrix,
th e acyl groups are tra n sferred back to coenzyme A (CoA) and oxidation can
th en occur. The oxidation of fatty acids occurs also in peroxisom es . 3 >4 >6 >7 The
oxidation in peroxisomes is not complete, b u t term inates a t acyl-CoA residues
of short- an d m edium -chain len g th . 3
These chain-shortened acyl-CoA
residues are th e n esterified to carnitine and th e resulting acylcarnitines are
tran sp o rted out of peroxisomes for fu rth er m etabolism.
A recent study 8
suggests th a t carnitine is involved in the m etabolism of both short- and longchain acyl-CoAs w ithin lymphocytes and phagocytes. C arnitine m ay play a
significant role in th e m etabolism of m edium -chain fatty acids as w ell . 9
In
addition, carnitine can buffer th e acylation state of the CoA pool in ways
specific to th e local m etabolic dem ands . 1 9
C arnitine is essential for good health.
concentrations
m etabolism . 1
of carnitine
Tissues w ith inadequate
exhibit severely im paired
cellular energy
A diverse collection of diseases are related to carn itin e . 1 -5
Since th e discovery of th e first case of h um an carnitine deficiency , 1 1 carnitine
has been used for th e ra p y . 1 2 Alzheimer's disease is the m ost common cause
of dem entia
in
developed
countries
and
affects
A m ericans . 1 3
The progression of Alzheim er's disease can be significantly
reduced by adm inistration of acetylcarnitine . 1 3
about
3-4
million
C arnitine also plays an
essential role in h u m an n u tritio n . 5 - 1 4 R equirem ent for carnitine is increased
by rap id grow th (infancy, or th e repletion of protein-calorie undernutrition),
pregnancy, lactation, metabolic acidosis, certain drugs excreted in acidic
form, ren al dialysis and ren al tu b u la r disorders . 1 4
C arnitine functions as an acyl carrier in fatty acid m etabolism and
acts as a buffer for th e acyl-CoA/CoA ratio.
The reversible acyl tran sfer
betw een carnitine and CoA is catalyzed by carnitine acyltransferases
3
acyltransferase
q
CoAS-
CoASH
O
N -M e
N —M e
/ v
Me
Me
Me
Me
carnitine
coenzym e A
S ch em e 1.1
(Scheme 1.1 ) . 2 " 4 ’ 1 5
Therefore, both carnitine an d carnitine acyltransferases
play vital roles in fatty acid m etabolism and are essential for good health.
C arnitine acetyltransferase (CAT), carnitine octanoyltransferase (COT), and
carnitine palm itoyltransferase (CPT) have been identified.
They are
classified on th e basis of th e ir subcellular localization an d su b strate
specificity . 2 CAT, which is selective for short-chain acyl groups, is found in
m itochondria, peroxisomes, an d m icrosom es . 1 6
m edium -chain/long-chain
microsom es . 1 ^ ' 2 1
acyl
groups
is
COT w ith specificity for
found
in
peroxisomes
and
CPT, selective for long-chain acyl groups, is found on the
m itochondrial outer m em brane (CPT-I) and in th e m itochondrial m atrix
(CPT-II).17>18>22-27
erythrocyte
CPT is also found in microsomes , 2 8 peroxisom es , 2 9 ’ 3 0
plasm a
m em brane , 3 1
and
sarcoplasm ic
reticulum . 3 2
Considerable overlap of acyl chain length selectivity occurs for COT and CPT.
O ur long-range goal is to elucidate th e stru ctu res of th e m olecular
interactions betw een carn itin e and the active sites on these enzymes.
Identifying these m olecular in teractions will lead to a b etter u n d erstan d in g
of th e physiological chem istry and, more specifically, of the regulation of the
enzymes. This project will focus on CAT.
1.2.
CARNITINE ACETYLTRANSFERASE
CAT is widely d istrib u ted in n atu re, occurring in different organism s
from yeast to m am m als . 2 Very high CAT activities are observed in r a t heart,
testis, an d brow n adipose tissu e . 3 3 CAT is also uniform ly distributed in the
nervous system . 3 4 - 3 5
CAT h as been purified from different tissues and organelles, including
pigeon
b re ast
m uscle
hom ogenate , 3 6 ' 3 0
pig
h e a rt , 4 0
mouse
liver
peroxisom es , 2 0 y east peroxisom es , 4 1 yeast m itochondria , 4 1 yeast cell-free
ex tract , 4 2 -4 3 r a t liver m itochondria , 4 4 -4 5 r a t liver peroxisom es , 4 6 r a t liver
m icrosom es , 4 6
ra t
liver
hom ogenate , 4 5
bovine
sperm atozoa , 4 7 chick embryo liv er , 3 0 and h u m an liver . 4 8
h e a rt, 31,41
bovine
In general, CATs
from different sources have sim ilar properties w ith m olecular w eight ranging
from 51,000 to 75,000 an d pH optim um betw een 7.2 and 8.0 . 1 6 M ost of the
CATs show th e h ig h est activities w ith C 3 or C 4 acyl groups . 1 6
The wide distrib u tio n of CAT indicates its im portant physiological
functions.
The role of CAT in buffering acetyl-CoA/CoA ratio in the
m itochondrial m atrix has been established . 1 0
In the m itochondrial m atrix,
free CoA is required for th e function of th e citric acid cycle, for p-oXidation,
for th e detoxification of organic acids, for th e oxidative degradation of amino
acids, and as a su b strate for pyruvate dehydrogenase and a-ketoglutarate
dehydrogenase.
Therefore, lack of free CoA can lim it th e m itochondrial
capacity for energy production. CAT can m odulate th e acyl-CoA pools and
facilitate form ation of free CoA u n d er conditions of accum ulating acyl-CoA.
In h u m an m uscle tissue, an increase in acetylcarnitine occurs during
endurance exercise , 4 9 *5 0 indicating th a t, u n d er the regulation of CAT,
carnitine functions as an acceptor for an acetyl group to buffer excess acetylCoA form ed from pyruvate decarboxylation and P-oxidation and release CoA
for energy production.
The m odulation of the acyl-CoA pools m ay also be
im p o rtan t in m etabolic regulation in norm al tissu e , 2 protecting the tissue
from build-up of toxic acyl-CoA 5 1 - 5 3 and providing a p ath w ay for sto rin g or
ex creting th e acyl m oieties if th e ir norm al m etabolism is im p a ire d . 2 -5 3 -5 4
A cetylcarnitine can serve as a reservoir of activated acetyl u n its and provide
an im m ediate source of energy . 2 - 1 0 CAT found in endoplasmic reticulum and
in peroxisomes can convert cytosolic acetylcarnitine into acetyl-CoA, which
could be used for th e synthesis of malonyl-CoA in the heart. Accum ulation of
acetyl-CoA in th e cytoplasm would also serve to decrease th e free CoA
available for fatty acid activation . 5 5 CAT in each organelle or m em brane can
m odulate th e reserves of free CoA and acetyl-CoA in ways specific to the local
dem ands . 1 0
The buffer role of CAT in general fits in well w ith th e tissue
distribution of th e enzyme, which correlates w ith high m etabolic activity . 5 6 -5 7
A nother im p o rtan t role for CAT is th a t it m ay sh u ttle
short-chain
acylcarnitines out of peroxisomes for tissues th a t chain-shorten long-chain
fatty acids via peroxisom al P-oxidation . 2 - 1 0
The functions of CAT indicate its im portant roles in hum an health.
The significance of CAT in h u m an h ealth was dem onstrated by the
discoveries th a t deficiency in CAT activity was p resen t in the b rain and other
tissues of a p a tie n t suffering from fatal ataxic encephalopathy 5 8 and in the
brain, h e a rt, and kidney of a baby w ith poor respiration, failure to thrive and
low levels of esterified carn itin e in u rin e . 5 9 CAT activity is decreased in the
b rains an d m icrovessels of p atien ts w ith A lzheim er's disease . 6 0
A large lite ra tu re exists about CAT. The advances in the area of CAT
up to 1987, including physical properties, kinetics, and inhibition of CAT,
have been review ed by Colucci an d G andour . 1 6 Several generalizations have
been m ade about th e CAT-catalyzed acetyl tra n sfer reaction betw een
carnitine an d CoA.
The binding of carnitine and acetylcarnitine probably
requires recognition of both carboxylate and q u atern ary am m onium portions
of th e molecule an d m ay involve recognition of charge. B inding of carnitine
occurs by an induced-fit m echanism and binding of CoA, or acetyl-CoA,
occurs in a lock-and-key fashion. The kinetic m echanism fits a random -order
equilibrium model. The m olecular m echanism of acetyl tra n sfe r is probably
addition-elim ination w ith general base catalysis of hydroxy attack on a
th ioester in th e forw ard reaction and general acid catalysis of oxygen
expulsion in th e reverse.
This section will sum m arize recent progress in the a rea of CAT
enzymology.
1.2.1. CATs from different sp ecies
Table 1.1 lists th ree CATs purified from different species in recent
years. The m olecular w eights of these enzymes are sim ilar to other CATs . 1 6
The resu lts of th e study of Bloisi et al 4 8 indicate th a t h um an liver CAT is a
monomer, sim ilar to mouse liver CAT . 2 0 The pH optim um for activty of CATs
from chick embryo liver an d yeast S. cerevisiae are
sim ilar to m ost of the
others, while th e pH optim um for activity of CAT from hum an liver is higher
th a n o th ers . 1 6
U nder pH 6.0, th e enzyme from S. cerevisiae undergoes an
irreversible inactivation. The su b strate specificity of th e enzyme from chick
embryo liver is sim ilar to th a t of CAT in pigeon b reast m uscle w ith m axim um
activity w ith acetyl group, b u t different from m ost of the others th a t utilize
C 3 or C4 acyl group as th e optim al s u b stra te . 1 6 The CAT from hum an liver
has th e h ighest activities w ith C 3 acyl groups, which is sim ilar to m ost of the
other CATs . 1 6
T able 1.1. CATs p u rified from d ifferen t species
Enzym e
source
M olecular
w eig h t
pH
optim um
Isoelectric
point
Ref.
H u m an liv er
60,500
8.7
6.3
C hick em bryo liv er
64,000
8 .0
30
Y east S. cerevisiae
65,000
7.5-8.0
43
48
Table 1.2 lists th e ap p a re n t K m values of CATs for different chainlengths of su b strates in th e forw ard and reverse reactions. The K m values
indicate th a t th ese enzym es bind CoA (or acetyl-CoA) more tightly th a n they
bind carnitine (or acetylcarnitine).
For th e CATs from chick embryo liver
and h u m an liver, th e K m values for carnitine also indicate th a t short-chain
acyl-CoAs are preferential su b strates w hen the concentration of carnitine is
low.
T able 1.2. V alues of K m of CATs for differen t s u b stra te s
K m (|iM)
R ef
Enzym e
source
S u b stra te
H u m an
liv er
c2
c3
c4
C6
C8
Cio
21.3
28.0
53.5
54.9
50.3
64.0
97
86
152
120
148
580
Chick
embryo
liver
c2
C4
C6
C8
52
130
66
94
160
250
1100
2000
S. cerevisiae
C2
17.7
170
acyl-CoA
carn itin e
CoA
acy lcarn itin e
420
650
48
1390
86
53
78
66
800
890
900
670
30
43
1.2.2. Am ino acid seq u en ces o f CATs
A cDNA encoding for in n er m itochondrial CAT of yeast S. cerevisiae
was isolated by screening a yeast cDNA X g tll lib rary w ith antibody . 6 1
The
whole coding sequence, which consists of 670 am ino acid residues w ith a
m olecular w eight of 77.3 kD a, was obtained from the cDNA and from a YEP
13 DNA clone identified usin g th e cDNA as probe.
The identity of this
isolated cDNA was confirmed by using it to d isru p t the yeast chromosomal
CAT gene.
The elim ination of CAT activity from the m itochondria of the
transform ed cells w as shown by m easuring CAT activity and by im m unoblot.
The acetylcarnitine content of these cells decreased significantly, indicating
th a t no other m ajor CAT activity rem ained in th e cells.
The residual low
CAT activity in th e cytosol of m u ta n t cells did not change, suggesting th a t
this cDNA codes for th e m itochondrial isoenzyme of CAT.
The gene YAT1 3 2 from S. cerevisiae encodes a protein of
6 8 8
am ino
acids w ith a calculated m olecular m ass of 77.9 kDa, which displays
significant sequence sim ilarity to vertebrate carnitine acyltransferases and
yeast in n er m itochondrial CAT. The researchers of th is study failed to prove,
by m u ta n t phenotype analysis, th e exact su b strate specificity of th e encoded
enzyme. Based on other evidences an d data, they suggest th a t th is novel
gene is likely to code for a m inor m itochondrial outer CAT.
1.2.3. In hib ition o f CATs
For a detailed review of inhibition of CAT up to 1987, see ref. 16.
I.2.3.I. CoA analogues
Table 1.3 lists three CoA analogues th a t in h ib it CATs from different
sources.
Vessey et al 6 3 have studied the effect of carboxylic acid xenobiotics and
th eir m etabolites on th e activity of carnitine acyltransferases and found th a t
benzoyl-CoA an d salicylyl-CoA are potent inhibitors of CAT. For th e forw ard
reaction, both benzoyl-CoA and salicylyl-CoA are competitive inhibitors
10
T able 1.3. CoA analogues
Name
Structure
E nzym e source
Ref.
A
Sigma, St. Louis, M O , U SA
63
CoA
Sigina, St. Louis, M O , USA
63
O
benzoy 1-CoA
^
C
o
salicy ly 1-Co A
O
purified rat liver peroxisom es
64
purified nit heart mitochondria
64
partially purified rat liver mitochondria 64
C oA
etom oxiryl-CoA
(CH2)— O
versus acetyl-CoA w ith K\ v alu es of
Cl
2 2
pigeon breast m uscle (commercial)
a n d 7.5 gM respectively.
64
For the
reverse reaction, both are competitive inhibitors versus CoA w ith K\ values of
17 an d 9.5 |_iM respectively. For comparison, they determ ined th e K\ for endproduct inhibition by CoA an d obtained the value of 22 |iM. Benzoyl-CoA has
about th e sam e K\ as CoA. Salicylyl-CoA has a lower K\ for the enzyme th a n
CoA or benzoyl-CoA.
The resu lts indicate th a t benzoate group has little
effect on binding. It is not clear why th e addition of a hydroxy group should
im prove th e interaction w ith th e enzyme.
Etomoxiryl-CoA is an o th er CoA analogue inhibitor th a t was tested
w ith CATs from different sources . 6 4 The d a ta are sum m arized in Table 1.4.
The concentrations of etomoxiryl-CoA required for 50% inhibition of the
different CATs are in th e low m icrom olar range.
11
Table 1.4. Inhibition of CATs by etomoxiryl-CoA
Enzyme source
Kj (pM)
Comment
Purified from rat liver peroxisomes
2
Mixed-type inhibition with acetyl-CoA
Purified from rat heart mitochondria
9
Mixed-type inhibition with acetyl-CoA
Partially purified from rat liver mitochondria 2
Mixed-type inhibition with acetyl-CoA
pigeon breast muscle (commercial)
Uncompetitive inhibition with acetyl-CoA
3
I.2.3.2. C arnitine an alogu es
S tudies of carnitine analogues w ith CAT have detailed the criteria for
m olecular recognition a t its active site . 1 6
Table 1.5 lists several carnitine
analogues th a t have been synthesized and assayed recently . 6 5 -6 6
HDH, Ac-
HDH, (f?!S)-3-amino-5,5-dimethylhexanoic acid, and (i?S)-A^-acetyl-3-amino5,5-dim ethylhexanoic acid are competitive inhibitors of pigeon b re a st CAT.
(f?)-(+)-HDH, (<S)-(-)-HDH, CR)-3-amino-5,5-dimethylhexanoic acid, and (S)-3am ino-5,5-dim ethylhexanoic acid are stereoselective competitive inhibitors of
CAT. None of them is a su b strate for CAT. These results indicate th a t the
positive q u atern ary am m onium charge on carnitine is essential for CAT
catalysis.
In order to determ ine the active conformation about C2-C3 of
carnitine, B rouillette et al 6 7 designed cyclic carnitine analogues which
contain defined spatial relationships betw een the q u atern ary am m onium ,
hydroxy, an d carboxylate m oieties (table 1.6). None of them is a su b strate
12
T a b le 1.5. Inhibition of CAT by carnitine analogues
Nam e
Structure
(± )-3-H ydroxy-5,5-dim ethylhexanoic acid (H D H )
CH3
(±}-3-A cetoxy-5,5-dim ethylhexanoic acid (A c-H D H )
CH3
(/?)-(+)-3-H ydroxy-5,5-dim ethylhexanoic acid (H D H )
CH3
(,S')-(-)-3-Hydroxy-5,5-dimethylhexanoic acid (H D H )
CH,
(/6S')-3-Am ino-5,5-dimethyI
hexanoic acid
(/6S’)-N -A eetyl-3-am ino-5,5dim ethylhexanoic acid
V alue ((J.M)
GA
CH,
(ft)-3-Aminc>-5,5-dimethyl
hexanoic acid
CH3
Pigeon breast
65
4 ,1 0 0
Pigeon breast
65
20 ,3 0 0
Pigeon breast
65
7 ,5 0 0
Pigeon breast
65
2 ,6 0 0
Pigeon breast
24,8 0 0
Pigeon breast
66
1,900
Pigeon breast
66
9,2 0 0
Pigeon breast
66
OH
OH
NH
NHAc
CO,
(,S')-3-Amino-5,5-dimethyl
hexanoic acid
8,300
c
CO,
CH3
Ref.
OH
CO,
CH3
Enzym e source
NH
NH2
,C O ,
13
for pigeon b re a st CAT a t concentrations up to 10 mM.
They are weak
competitive inhibitors of CAT. The K\ values are m uch larg er th a n the K m
for CR)-carnitine (300 pM) or (R )-acetylcarnitine (300 pM). The resu lts reveal
th a t these analogues bind m uch less tightly th a n carnitine or acetylcarnitine
to CAT.
The possible reaso n s 6 7 for the resu lts are th a t (1) none of the
conformations of th e analogues represents the bound conformation for
carnitine or acetylcarnitine, or (2 ) the extra steric bulk provided by the
cyclohexyl rin g residues in terferes w ith binding.
Table 1.6. Inhibition of CAT by rigid carnitine analogues
C om p ou n d (racem ate)
NMe.
R
(p M )
H
4 ,1 0 0
Ac
3 ,7 0 0
H
2 ,9 0 0
Ac
10,700
H
15,900
Ac
2 3 ,0 0 0
OR
NMeOR
H
NMe.
OR
14
I.2.3.3. B ile acids
Bile acids have different inhibitory effects on CAT (Table I.7 ) . 6 8 -6 9
W hile determ ining th e concentration of carnitine in r a t bile, Sekas and
P a u l 6 8 have observed th a t th e concentration increased progressively as the
bile was diluted, which prom pted them to study inhibition of bile acids on
T able 1.7. Bile acids as in h ib ito rs of CAT
N am e
S truct ure
E n zy m e source
Ref.
purified pigeon breast m uscle
68
purified pigeon breast m uscle
68
purified pigeon breast m uscle
68
purified pigeon breast m uscle
68
rat liver peroxosom es (in vitro)
69
HO
COOH
cholic acid
OH
HO
COOH
deoxycholic acid
COOH
lithocholic acid
H O ....
COOH
chenodeoxy cholic
acid
H O '""
OH
15
CAT. They have exam ined inhibitory effects of m ajor bile acids found in bile.
All bile acids tested (Table 1.7) in h ib it CAT to varying degrees.
At
physiological concentrations, th e degree of inhibition is chenodeoxycholic >
cholic > deoxycholic > lithocholic acid. For the forward reaction, K j value for
chenodeoxycholic acid is 520 pM.
The inhibition is competitive w ith
carnitine. The reverse reaction is inhibited by 25, 30, 63, and 89% in the
presence of 0.23, 0.45, 0.90, an d 1.6 mM chenodeoxycholic acid, respectively.
They have also studied inhibition of CAT by bile acids in r a t liver
peroxisom es . 6 9 The a p p aren t K\ value is 890 pM. Inhibition is observed both
in vitro and ex vivo.
I.2.3.4. C onform ationally con strain ed reaction -in term ed iate
analogu es
This type of inhibitors is the m ost powerful tool in probing the
stru ctu re of enzyme binding sites while they are in the conformations
adopted for catalysis. Several of this type of inhibitors have been synthesized
an d te ste d . 1 6
Table 1.8 lists three analogues th a t have been tested
recently . 7 0 Table 1.9 lists th e ir inhibition constants w ith various CATs. All
these hem iacylcarnitinium s are competitive inhibitors.
HAC binds better
th a n CR)-carnitine, (R )-acetylcarnitine, or CoA to CAT. HBC w eakly inhibits
CATs. It binds to CATs less tightly th a n do the substrates. The degree of
inhibition is HAC > H PrC > HBC due to the different substitution groups on
C2 (methyl, ethyl, and phenyl group). Nonracemic HAC inhibits CAT more
16
strongly th a n racem ic HAC , ? 1 indicating the im portance of configurations of
C 2 an d C 6 in enzyme recognition.
Table 1.8. Conform ationally constrained reaction-interm ediate analogues
Name
(2S,<5ft)-6-(Carboxylatomethyl)2-hydroxy-2,4,4-trimethylmorpholinium
(hemiacetylcarnitinium, HAC)
Structure
H
CH
-O
Me
(2S,6/?)-6-(Carboxylatomethyl)2-ethyl-2-hydroxy-4,4dimethylmorpholinium
(hemipropanoylcarnitinium, HPrC)
OH
Me
H
OH
N
Me'
(2S,h/?)-6-(Carboxylatomethyl)2-hydroxy-4,4-dimethyl-2phenylmorpholinium
(hemibenzoylcarnitinium, HBC)
H
Me
OH
Ph
Me
Me
The inhibitory activity of HAC is explained by its stru ctu ral sim ilarity
to th e tetrah ed ral-in term ed iate proposed in th e acetyl tra n sfer betw een CoA
and acetylcarnitine (Figure I . l ) . 7 1 >7 2
17
Table 1.9. Inhibition constants of hem iacylcarnitinium s w ith CAT
Am(pM)
Inhibitor
Ai(|xM)
(R )-Acetylcarnitine 290 + 20
(-R)-Carnitine
143
CoA
130 ±20
HAC
69 ± 5
Pigeon breast
(R)-Carnitine
1 2 0
(i?)-Acetylcarnitine 350
HPrC
200 ±30
Pigeon breast
Rat heart mitochondria
Rat liver peroxisomes
(-R)-Carnitine
1 2 0
CoA
1 2 0 ± 1 0
(Rl-Acetylcarnitine 280 ± 10
HBC
3500 ± 500
2400 ± 70
1670 ± 70
Enzyme source
Substrate
Rat liver peroxisomes
Rat heart mitochondria
92 ± 14
C oA
CH
O.
Me
Me
Me
Me
F ig u re 1.1. S tru c tu ra l re la tio n sh ip betw een th e
proposed te tra h e d ra l-in te rm e d ia te in th e acetyl
tra n s fe r reactio n a n d th e s tru c tu re of HAC
I.2.3.5. O ther in h ib itors
M ethylglyoxal bis(guanylhydrazone) (MGBG) inhibits purified pigeon
b re a st CAT w ith K\ value of 1.6 m M . 7 3
It is competitive w ith CR)-carnitine.
The com petitive inhibition of MGBG w ith c a rn itin e is probably due to the
s tru c tu ra l sim ila rity betw een MGBG a n d c a rn itin e or acetylcarnitine as
proposed by B rady et a l . 7 3
The guanidium group occupies the q u atern ary
am m onium site an d th e chain extends in a sim ilar fashion to carnitine w ith
an im ine group occupying a position sim ilar to th e acyl carbonyl.
In this
location, MGBG can form a thioam inal w ith eith er CoA or cysteine p resen t in
th e active site.
NH
CH 3 ^ ^ N “ N H “ Ct
C
nh2
NH
nh
2
MGBG
H um an liver CAT4® an d chick embryo liver CAT®^ are inhibited by
Ca2+ which doesn't in h ib it other avian and m am m alian CATs.
16
Mouse liver CAT is inhibited by alachlor, a herbicide . 7 4
^CCH9C1
Ns.
c h 2o c h 3
alachlor
1.2.4. Sum m ary
Some ideas are fu rth e r confirmed. CAT enzym atic catalysis requires a
positive q u atern ary am m onium charge on carnitine.
The chiralities of
(acetyl)carnitine and th e proposed tetrahedral-interm ediate are im portant
for CAT catalysis.
19
1.3.
DESIGN OF INHIBITORS OF CARNITINE
ACYLTRANSFERASE
The c a rn itin e a c y ltra n sfe ra se s are a fam ily of enzym es th a t differ
w ith resp ect to su b c ellu lar location, s u b stra te specificity, a n d sen sitiv ity
to in h ib ito rs . 2
T hey catalyze acyl tra n s fe r betw een c a rn itin e an d CoA
(Schem e 1.2).
The th re e-d im en sio n al s tru c tu re s of th e se p ro te in s are
unknow n.
= J)H
S -M e
/ '
Me
A c y ltra n sfe ra se
+
►
CoAS— p— R
O
q
+
O
4 \+
*
N —M e
/ \
Me
Me
C oA S H
Me
c o en z y m e A
carnitine
S ch em e 1.2
In o rder to probe th e s tru c tu re s of th e m olecular in tera ctio n s
betw een c a rn itin e a n d th e active sites in th ese enzym es, we have designed
conform ationally co n strain ed an alo g u es to m im ic th e proposed reactionin te rm e d ia te for acyl tra n s fe r betw een c a rn itin e a n d CoA. By com paring
in h ib itio n co n sta n ts (ATj's) of a series of conform ationally co n strain ed
analogues, we can id en tify th e topographical a rra n g e m e n t of th e key
recognition sites on th e se enzym es.
1.3.1. C o n sid era tio n s in th e en zy m e in h ib ito r d e sig n
S everal
co n sid eratio n s
m u st
be
m ade
w hen
designing
conform ationally co n strain ed re actio n -in term ed iate analogue in h ib ito rs.
20
F irs t, we m u st consider th e possible enzym e recognition sites in th e
su b stra te s.
The
enzym es,
am m onium , carboxylate,
especially
an d
CAT , 7 5
acyl group
recognize
trim ethyl-
on acylcarnitine.
R ecent
stu d ie s 6 5 - 6 6 su g g est t h a t th e positive q u a te rn a ry am m onium charge on
ca rn itin e is esse n tia l for CAT catalysis. The enzym es also recognize CoA.
Second, th e conform ations of th e s u b stra te s m u st be considered.
T here a re n in e possible conform ations for carn itin e .
To design a
conform ationally co n stra in ed analogue, we need to know w hich of th e
nin e
conform ations
of c a rn itin e
is
bound
to
th e
enzym e.
The
conform ational an aly ses of c a rn itin e a n d acety lcarn itin e have been
perform ed
by
u sin g
single
cry stal
m olecular m echanics (M M 2 ) . 7 6
X -ray
diffractio n , 7 2
NMR,
and
Those s tru c tu ra l s tu d ie s 7 2 - 7 6 show th a t
c a rn itin e a n d a c e ty lca rn itin e strongly p refer gauche (-) conform ation
ab o u t N -C 4-C 3-0 to rsio n angle.
B ased
on
th e se
conform ational
an aly ses
of
c a rn itin e
and
acety lcarn itin e, we can lock th e N -C 4-C 3-0 torsion angle in th e gauche (-)
conform ation by fo rm atio n of a rin g an d probably n o t lose m uch in
b in d in g to th e enzym e. T hese analogues m u st have groups n ecessary for
enzym e
recognition
an ch o red
to
th e
conform ationally
constrained
m olecular fram ew ork in a w ell-defined stereochem istry.
T hird, th e enzym e in h ib ito rs should m im ic th e s tru c tu re of a
re a ctio n -in te rm ed ia te
or
a
tra n s itio n
s ta te
of
th e
m echanism .
W olfenden 7 7 h a s pio n eered th e developm ent of tra n sitio n -s ta te analogue
21
in h ib ito rs of enzym es.
The idea is th a t enzym es b in d tra n sitio n
stru c tu re s or reac tio n -in term e d iates m ore tig h tly th a n re a c ta n ts
products.
or
M olecules th a t resem ble th e s tru c tu re s of tra n sitio n s ta te s or
rea ctio n -in te rm e d ia tes, b u t a re u n reactiv e, will b in d strongly to th e
enzym es. A m echanism for acetyl tra n s fe r in CAT h a s been proposed 7 2
(Schem e 1.3) b ased on chem ical model stu d ies of O-to-S acyl tr a n s f e r . 7 8 I t
is p resu m ed t h a t a sim ilar m ech an ism operates in COT an d C PT . 7 9
coas
;
O.
/
Me
N -M e
'N -M e
\
\
Me
Me7 M e
tetrahedral intermediate
C oAS
/ \
Me
Me
S ch em e 1.3
In form ing such a te tra h e d ra l-in te rm e d ia te , th e th io la te should
ap p ro ach th e
acyloxy from th e less-congested
side (carboxylate on
ca rn itin e "folded" back). T his a tta c k arrow is on th e Re face of th e ester,
p re su m in g th a t th e acyloxyl group is in th e m ost stab le conform ation . 7 6
T his R
configuration
of th e
te tra h e d ra l-in te rm e d ia te
can
ad o p t
a
22
conform ation t h a t favors in tra m o le c u la r electro static c ataly sis.80
The
developing neg ativ e charge on th e carbonyl oxygen w ould be stab ilized by
th e positively charged trim eth y lam m o n iu m group.
T he enzym es also
recognize th e configuration a t C3 of (acyl)carn itin e d u rin g th e acyl
tra n sfe r.
A rea ctio n -in te rm ed iate analogue m u st m im ic b o th of th ese
configurations.
1.3.2. D e sig n o f r ea c tio n -in te r m e d ia te a n a lo g u es
C onform ationally co n strain ed analo g u es are th e m ost effective for
exam in in g th e topography of a n active site in a n enzym e w hen catalysis
occurs.
B ased
on th e
(acyl)carnitine,
th e
stu d ies
on
conform ational
th e
possible
stu d ies
on
b in d in g
sites
c a rn itin e
in
and
acety lcarn itin e, an d th e an a ly sis of th e possible m echanism for acyl
tra n sfe r, we hav e designed th e re a ctio n -in term ed iate an alo g u es as shown
in F ig u re 1.2.
The m orp h o lin ium rin g adopts a c h a ir conform ation.
T herefore, th e N -C 5-C 6-0 to rsion angle in th e m orpholinium rin g is
S C oA
H
I —R
H
Y
Y = H
OH
/ '
Me
C H -C o A
Me
tetrahedral intermediate
/
Me
\
Me
ttntilogue inhibit or
F ig u re 1.2. Proposed te tra h e d ra l-in te rm e d ia te a n d analogue
in h ib ito r
23
locked in a gauche (-) conform ation, w hich is sim ilar to N -C 4-C 3-0 torsion
angle in (acyl)carnitine. The carboxym ethyl an d th e alkyl ch ain (R) are
cis d ieq u ato ria l in th e m ost stab le conform ation. A covalent bond (C2-C3)
in th e m orpholinium rin g replaces th e electro static in te ra c tio n betw een
th e neg ativ ely charged carbonyl oxygen an d th e positively charged
trim eth y lam m o n iu m group in th e reactio n -in term ed iate. A six-m em bered
rin g w as chosen because of ease of sy n th esis a n d few er conform ations.
S everal conform ationally co n strain ed cyclic analogues, m ade in th is
lab o rato ry ,
ex h ib it
in h ib ito ry
activities
tow ard
d ifferen t
c arn itin e
a c y ltra n sfe ra se s . 1 6 >7 0 >7 9 >8 1 F or exam ple, (+ )-hem iacetylcarnitinium (HAC,
R = CHg, Y = OH, F ig u re 1.2) in h ib its r a t liv er peroxisom al CAT a n d r a t
h e a r t m itochondrial CAT ; 7 0
(+ )-hem ipalm itoylcarnitinium (HPC, R =
(CH 2 ) i 4 C H 3 , Y = OH) in h ib its m itochondrial CPT-II a n d peroxisom al COT
of r a t liv e r , 7 9 an d r a t h e a r t an d r a t liver m itochondrial C PT -I . 8 1
In th e se analogues, th e location of Y (OH) is w here CoA a tta c h e s to
th e proposed reactio n -in term ed iate.
In ord er to te s t th is id ea an d
in crease th e potency of th e in h ib ito rs, we need a p la n to synthesize
an alo g u es w here Y = CH 2 -C 0 A.
1.4.
PROJECT GOAL
The u ltim a te goal of th is project is to synthesize an analogue, I, of
th e
proposed
te tra h e d ra l-re a c tio n -in te rm e d ia te
b etw een ca rn itin e an d CoA.
for
acetyl
tra n s fe r
T his analogue contains both c a rn itin e and
24
2
nh
OH
'()—P—O—P—O
,M e
OH
OH
OH
Me
Me
P—O'
II
O
I
CoA frag m en ts (Y = C H 2 -C 0 A); those previously synthesized contain only
th e c a rn itin e frag m en t a n d hydroxy group (Y = OH). The m orpholinium
rin g in th is an alo g u e co n tain s two ch iral centers, so th e re are four
stereoisom ers. The previously synthesized analogue in h ib ito rs are e ith e r
racem ic m ix tu re or only one stereoisom er. The lab ility of th e hem ik etal
lin k ag e
allow s
co n fig u ratio n . 7 0
th e
carbon
to
eq u ilib rate
to
th e
m ore
stable
In th e se m olecules, hydroxy group prefers to be axial.
The goal of th is project is to synthesize all th e four stereoisom ers of the
re ac tio n -in term ed iate analogue.
By com paring th e in h ib ito ry effects of
th ese four stereoisom ers, m ore inform ation about th e topographical
a rra n g e m e n t of th e key recognition sites on th e enzym es can be obtained.
T his in fo rm atio n will lead to a three-d im en sio n al p ictu re of th e active
sites of th e enzym es.
25
1.5.
SPECIFIC GOAL
The goal of m y resea rc h is to develop sy n th etic stra te g ie s for four
stereoisom ers of 6-carboxylatom ethyl-2-hydroym ethyl-2, 4, 4 -trim ethyl
m orpholinium , II, w hich are th e key sy n th etic in te rm e d ia te s in th e
proposed
ro u te
to
th e
four
stereoisom ers
of
2-(3-am inopropyl)-6-
carb oxylatom ethyl-2,4,4-trim ethylm orpholinium , III, w hich will serve as
p recu rso rs to I. The assay of those synthesized enzym e in h ib ito rs by Drs.
N oirfn N ic a B h aird an d R ena R. R am say a t U CSF is also a p a r t of th is
work.
/
Me
'
II
Me
/
Me
\
m
Me
Chapter
II
SYNTHESES OF INHIBITORS OF
CARNITINE ACETYLTRANSFERASE
II. 1. RE TRO S YN THE TIC ANALYSIS
The syntheses of th e ta rg et molecules can be approached from
different pathw ays.
H erein, we exam ine the structures of all th e targ et
molecules w ith different retro synthetic strategies to analyze the possible
pathw ays to approach these molecules.
Both th e ta rg e t molecules II and III were disconnected into two m ain
components: m ethyl 4-brom o-2-butenoate and P-hydroxyamine, which carries
different functional groups for II and III and can be m ade in different ways
(Scheme II.1).
Me
Me
Y
Y = H, Me
X = OH
c h 2c h 2n h 2
Z = OH
c h 2c n
S c h e m e II. 1
26
Me
II. 1.1. Target m olecu le II
Two retro synthetic routes were proposed for synthesizing targ et
molecule
II,
6-carboxylatom ethyl-2-hydroxym ethyl-2,
4,
4-trim ethyl
m orpholinium , as shown in Schem e II. 2 .
'o,c
OH
Me
Me
Me
n
R oute B
^O H
OH
,M e
R oute A
HOv p
Br
Me
/ \
Me
6
O,
OH
OH
OH
,Me
HO.
+
HN
I
Me
Me
3
4
Schem e II.2
In retrosynthetic route A, m ethylation of th e te rtia ry am ine, m ethyl 2(6-hydroxym ethyl-4,6-dim ethylm orpholinyl)acetate, 2, followed by hydrolysis
of m ethyl ester
disconnected into
would give th e ta rg e t molecule II.
two p arts:
Compound 2 is
m ethyl 4-bromo-2-butenoate, 3, and
3-
28
m ethylam ino-2-m ethylpropane-l,2-diol, 4.
Compound 3 is commercially
available. Compound 4 can be m ade by rin g opening of 2-methylglycidol, 5,
w ith m ethylam ine. Compound 5 is commercially available for both R and S
configurations.
Ring closure of 3 w ith (R )-4 or (S)-4 would give a
diastereom eric m ixture of 2 , which could be separated by chrom atography.
In
retrosynthetic
route
B,
m ethyl-2,4,4-trim ethylm orpholinim n,
2-hydroxymethyl-6-(methoxycarbonyl)1, is disconnected into 3 and 3-
dim ethylam ino-2-m ethylpropane-l,2-diol,
6
, which could be m ade from (R)-5
an d (S)-5 by ring opening w ith dim ethylam ine. In this route, m ethylation of
te rtia ry am ine is not needed. So it seems shorter th a n route A. B ut ring
closure of 3 w ith (R)-6 or (S ) - 6 would give a diastereom eric m ixture of
q u a tern ary am ine salts,
th a n te rtia ry am ine,
2
1
, which m ight be m uch more difficult to separate
.
II.1.2. T a r g e t m o le c u le I I I
Schemes II.3, II.4, an d II.5 show the retrosynthetic routes for
synthesizing ta rg e t molecule III, 2-(3-aminopropyl)-6-carboxylatomethyl2 ,4,4-trim ethylm orpholinium .
In retrosynthetic route C (Scheme II.3), m ethylation of te rtia ry am ine
of m ethyl 2-[6-(2-cyanoethyl)-4,6-dimethylm orpholinyl]acetate,
8
, followed by
hydrolysis of m ethyl ester an d reduction of cyano group would give th e target
molecule III.
Compound
8
is disconnected into 3 and 5-methylamino-4-
hydroxy-4-m ethylpentanenitrile, 9, which could be m ade by
conversion of
29
th e hydroxy group of 5 into a cyanom ethyl group and rin g opening of the
epoxy w ith m ethylam ine.
.NH,
,M e
Me
Me
in
R o u te D
M e () 2C
,Me
CN
CN
R o u te C
Me
/
Me
N
\
10
OH
CN
CN
,Me
*
"T
HN
I
Me
Me
9
8
S c h e m e II.3
In
retrosynthetic
route
D
(Scheme
II.3),
2-(2-cyanoethyl)-6-
(m ethoxycarbonyl)m ethyl-2,4,4-trim ethylm orpholinium , 7, is disconnected
into 3 and 5-dim ethylam ino-4-hydroxy-4-m ethylpentanenitrile, 10, which
could be m ade th e sam e way as compound 9 using dim ethylam ine in place of
m ethylam ine. Ring closure of 3 w ith 10 would give a diastereom eric m ixture
30
of q u atern ary am ine salts 7, which m ight be m uch more difficult to separate
th a n compound 8 .
Scheme II.4 shows other retrosynthetic routes for m aking 10 and 9.
Compounds
10
an d
9
could
be
m ade
by
rin g
opening
of
3-(l-
m ethyloxiranyl)propanenitrile, 11, w ith dim ethylam ine an d m ethylam ine
respectively. Compound 11 could be derived from 3-m ethyl-3-buten-l-ol, 12,
which is commercially available. In these two routes, we have to resolve 11
or 10 an d 9.
HN
Me
9
Schem e II.4
In Scheme II. 5, th e ta rg e t molecule III could be derived from II or 2,
which could be m ade according to Scheme II. 2.
31
11.1.3. S u m m a ry
For ta rg e t molecule II, both routes A and B originate from the same
startin g m aterials, (R )- an d (S)-2-methylglycidol, 5, which are commercially
available. C hiral compounds (R )- and (S)-3-m ethylam ino-2-m ethylpropaneI,2-diol, 4, an d (R)- an d (S)-3-dim ethylam ino-2-m ethylpropane-l,2-diol,
6
,
could be derived from these two chiral startin g m aterials. The ring closure
products of m ethyl 4-brom o-2-butenoate, 3, w ith diols 4 or
6
would be
m ixtures of diastereom ers 2 an d 1, respectively. The diastereom eric m ixture
of q u atern ary am ine salts,
1
, would be m uch more difficult to separate th a n
th e diastereom eric m ixture of te rtiary am ines, 2. Therefore, route A would
be b etter th a n route B.
For ta rg e t molecule III, both routes C an d D originate from the same
startin g m aterials. In route D, we would have th e sam e problem as th a t in
Scheme
II. 2, route B for targ et molecule
II — the
separation
of
diastereom eric m ixture of q u ate rn ary salts. In th e routes shown in Scheme
II.4,
we
need
to
resolve
3-(l-m ethyloxiranyl)propanenitrile,
11,
5-
32
m ethylam ino-4-hydroxy-4-m ethylpentanenitrile, 9, or 5-dim ethylamino-4hydroxy-4-m ethylpentanenitrile, 10.
If route A in Scheme II.2 for targ et
molecule II works well, th e routes in Scheme II.5 for ta rg e t molecule III
m ight work.
By routes C or D, we can build up the side chain (cyanomethyl) before
th e ring-closure step, which would have lower yield th a n th e other steps
because
of potential
side reactions and
chrom atography to
separate
diastereom ers. Therefore, we chose to work on routes C and D before A and
B. Compound 5 is not optically p ure and needs optical enrichm ent. B ut after
th e optical enrichm ent by converting it into 4-nitrobenzoate ester followed by
recrystallization, it is very difficult to get it back. We have tried th e route in
Scheme II.4. Racemic 10 can be m ade from 12 and can be resolved as the
3,5-dinitrobenzoate esters by HPLC using a chiral column. For preparative
scale, th is resolution m ethod is tedious and tim e consuming. Therefore, we
have sw itched to routes A and B.
II.2. RESULTS AND DISCUSSION
O ur synthetic efforts can be divided into two parts:
1) syntheses of (2R,6R)-, (2S,6S )-, (2R,6S )-, and (2S,6R)-Qcarboxylatom ethyl-2-hydroxym ethyl-2,4,4-trim ethylm orpholinium , II.
2
) syntheses of m ethyl (2S,6S]2R,6R)- and (2S,6R;2R,6S)- 2-[6-(2-
cyanoethyl)-4,6-dim ethylm orpholinyl]acetate,
8
.
33
II.2.1. S yn th eses o f (2R,6R)-, (2 S ,6 S )-, (2R,6S)-, and (2S,6R)-6carboxylatom ethyl-2-hydroxym ethyl-2,4,4trim ethyhnorpholinium , II
The
syntheses
of four
stereoisom ers
hydroxym ethyl-2,4,4-trim ethylm orpholinium ,
of
6
ta rg e t
-carboxylatom ethyl- 2 -
II,
molecule
are
illu stra ted in Scheme II. 6 .
CO., M e
1.
O ,. /
i X ^ O H
Cl-PNB
C H ^ C L /E t.N
-------- 2 - 3 -------| X
2.
Oh /
.O P N B
H ,N C H 3
recrystallization
(A’)-5
H
.N .
— 2---------------- 2 *
C H 3OH
sv'O H
.O H
^
Br
K ,C 0 3 /T H F
(S)-13
COOM
\
(.S')-4
COOMe
COOMe
O.
1. D B U /T H F W
OH
.... "
►
2. column
chromatography
(2S, 6S)-2
(2/\, 6S)-2
COOMe
OH
COOMe
C OO
CHd
NaOH
E t.,0
H .,0
1
(2R, 6S)-2
(.2S , 6R )-1
(2S, 6R)-U
Schem e II.6
According to the retrosynthetic analysis in Scheme II.2, (R)- and (<S)-3m ethylam ino-2-m ethylpropane-l,2-diol, 4, could be m ade from OS)- and (R)-2methylglycidol, 5, respectively by ring opening w ith m ethylam ine; (R)- and
34
(S)-3-dim ethylam ino-2-m ethylpropane-l,2-diol,
an d
(R)-2-methylglycidol,
dim ethylam ine
5,
respectively
6
, could be m ade from (S )by
rin g
opening
w ith
Compound 5 is commercially available for both R and
configurations.
Before startin g our synthesis, we m easured the optical purities of (inl­
and (S )-5 by iH NMR analysis of th e ir M osher's e ste rs . 8 2
We found th a t
th e ir optical purities w ere only about 90%. We needed the optical purities of
our compounds to be g reater th a n 97%.
Therefore, th e first step in the
synthesis was optical enrichm ent of 5. Gao et a l 8 3 showed th a t the optical
p u rity of 5 can be im proved by recrytallizing th e 4-nitrobenzoate ester. As
shown in
Scheme II. 6 , compound 5 was readily converted into
(1 -
m ethyloxiranyl)m ethyl 4-nitrobenzoate, 13, by tre a tm e n t w ith 1 equivalent
of 4-nitrobenzoyl chloride in dichlorom ethane in the presence of
equivalents of triethylam ine a t 0 °C.
1 .1
Successive recrystallizations from
ethanol, diethyl ether, an d isopropyl eth er gave optically enriched ester 13 in
49% yield.
The optical p u rity of 13 was determ ined by l-H NMR analysis (Scheme
II.7). Compound 13 was converted into
/
H N M e,
L > \ ^ 0PNB
|
V
sO H
by reaction w ith 4 equivalents of
C l-M T P A
'
V * ' 011 ~
*
► ^N^X^O M TPA
M eO H
13
6
E t3N /C H 2C l2
6
S c h e m e II. 7
15
35
dim ethylam ine in m ethanol. In this step, two reactions occur, ring opening
of epoxide an d cleavage of 4-nitrobenzoate, giving
6
. The product of the 4-
nitrobenzoate ester cleavage was m ethyl 4-nitrobenzoate in stead of N,Ndim ethyl 4-nitrobenzoate amide. This ester was removed by concentration of
th e reaction m ixture followed by filtration. Then compound
into
M osher's
e ster
15
by
tre a tm e n t
w ith
(trifluorom ethyl)phenylacetyl chloride (MTPA-C1).
6
was converted
(R)-{+)-a-m ethoxy-a-
!H NMR analysis of 15
indicated th e optical p u rities of both (R )- and GSO-13 g reater th a n 97% (the
m inor isom er w as undetectable).
Compound
6
was characterized by
NMR, !3C NMR, FT-IR, m ass spectroscopy, and elem ental analysis.
A fter th e optical enrichm ent, compound 5 could be recovered by
hydrolysis of 13.
(Scheme II. 8 ).
T hen rin g opening of 5 w ith m ethylam ine would give 4
So, two steps would be needed to convert 13 into 4.
We
th o ught th a t th e ester bond of 13 could be cleaved by am inolysis w ith
m ethylam ine, ju s t as it w as from 13 to
6
w ith dim ethylam ine, so th a t the
cleavage of th e ester bond an d ring opening of epoxy m ight take place in one
step. This way, we could save one step.
.OPNB
(S)-I3
►
{R)-5
h 2n c h 3/ c h 3o h
S c h e m e II. 8
(S)-4
36
Based on this idea, we tried the reaction of 13 w ith m ethylam ine
(Scheme II. 8 ). A solution of 13 in m ethanol was added into a solution of
6
equivalents of m ethylam ine in m ethanol a t room tem perature and the
m ixture was stirred for 3 hours. A-M ethyl 4-nitrobenzoate am ide produced
from the reaction w as insoluble in m ethanol and was removed by
concentration of the reaction m ixture followed by filtration. Compounds (R)an d (£>)-4 were obtained from (R)- and (S)-13 respectively in 75% yield and
characterized by Bd NMR, 13C NMR, FT-IR, m ass spectroscopy, and
elem ental analyses.
(.R)- and (S)-Methyl 4-[methyl-(2,3-dihydroxy-2-methylpropyl)amino]2-butenoate, 14, were prepared by reaction of (R)- and (S)-4 w ith 1
equivalent of m ethyl 4-brom o-2-butenoate in the presence of potassium
carbonate in THF a t room tem p eratu re overnight (Scheme II.9). At least 1
equivalent of potassium carbonate was needed to neutralize hydrogen
brom ide produced from th e reaction.
precipitate was removed by filtration.
After completion of th e reaction, the
Concentration of the solution gave
crude product 14 in 83% yield, which was used for next reaction w ithout
C O ,M e
(S)-4
COOMe
(S)-14
S c h e m e II.9
OH
37
fu rth er purification. From *H NMR spectra of the crude product, we could
see th a t th e reaction was a little cleaner in TH F th a n in diethyl ether.
Compound 14 w as characterized by 1H NMR, 13C NMR, FT-IR, and m ass
spectroscopy.
Ring
closure
of
14
in
THF
was
catalyzed
by
1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU)8^ to give m ethyl 2-(6-hydroxymethyl4,6-dim ethylm orpholinyl)acetate,
separated
by
column
2
, as a diastereom eric m ixture which was
chrom atography
(silica
gel,
230-400
mesh,
hexanes:dichlorom ethane:ethanol = 100:100:30) to give two diastereom ers
w ith a ratio of 3:1 an d to tal isolated yield of 40% (Scheme 11.10).
In this
reaction, 6 -m em bered ring w as formed instead of 7-mem bered ring because 6 m em bered rin g is more stable and easily formed th a n 7-membered rin g . 8 5
The m ajor isom er was th e one w ith the (methoxycarbonyl)m ethyl and
hydroxym ethyl adopting trans positions. From (<S)-14, we got (2R ,6S)- and
(2S,6S)-2.
From (R)-14, we got (2S,6R)- and (2R,6RY 2.
The four
stereoisom ers of 2 were characterized by !H NMR, 13C NMR, FT-IR, m ass
spectroscopy, and elem ental analyses.
COOMe
OH
COOMe
OH
COOM e
2. cnlum n
c h rom atography
CO-14
(2/?, 6.S')-2
Schem e 11.10
(2S, 6.S')-2
OH
38
The resu ltin g stereoselectivity m ay be attrib u te d to chair tran sitio n
stru ctu res in th e rin g closure reaction (Figure II. 1). The tra n sitio n structure
on th e rig h t is more stable an d easily formed th a n th a t on the left due to the
in tram o lecu lar in teractio n betw een the hydrogen of hydroxy group an d the
nitrogen. Therefore, th e tra n sitio n structure on th e rig h t is favored an d the
m ajor isom er of th e product is th e one w ith th e (m ethoxycarbonyl)m ethyl and
hydroxym ethyl adopting trans positions.
H— O
OH
Me
OH
M e-----N
Me
N
5*
COoMe
OH
M e S+
F ig u r e II. 1. T ran sitio n stru ctu res for ring closure reaction of 14
T reatm en t of
tem p eratu re
2
w ith excess of iodom ethane in diethyl eth er a t room
gave
2-hydroxym ethyl-6-(methoxycarbonyl)methyl-2,4,4-
trim ethylm orpholinium iodide, 1, as a light yellow solid in 74% yield.
At
le ast 15 equivalents of iodom ethane were needed for the completion of the
reaction. C rystals for X-ray analysis were obtained by recrystallization from
m ethanol by vapor diffusion w ith diethyl ether. The four stereoisom ers of 1
were characterized by *11 NMR, 13C NMR, FT-IR, m ass spectroscopy, and
elem ental analyses, an d verified by single-crystal X-ray analyses (Figure II.2
an d II.3).
39
In crystals, th e m orpholine ring of 1 is in a chair conformation. For
(2R,6S)-1 an d its enantiom er (2S,6R)-1, the (methoxycarbonyl)m ethyl and
m ethyl on C3 are cis ; both occupy equatorial positions. F or (2S,6S)-1 and its
enantiom er (2R,6R)- 1, th e m ethoxycarbonylm ethyl and hydroxym ethyl are
cis; both occupy equatorial positions. The (methoxycarbonyl)m ethyl alw ays
occupies an equatorial position for all four stereoisomers.
T reatm en t of 1 w ith
1
equivalent of sodium hydroxide solution (0.1 N)
gave th e ta rg e t molecule II, 6-carboxylatomethyl-2-hydroxymethyl-2,4,4trim ethylm orpholinium , which was recrystallized from m ethanol/acetone
(1/11) as colorless crystals in 67% yield. The four stereoisom ers of II were
characterized by !H NMR, 13C NMR, FT-IR, m ass spectroscopy, and
elem ental analyses, an d verified by single-crystal X-ray analyses (Figure II.4
and II.5).
In crystals, th e m orpholine ring of II adopts a chair conformation. For
(2R,6S)-I1 an d its enantiom er (2S,6R)- II, th e carboxylatom ethyl and m ethyl
on C3 are cis; both occupy equatorial positions.
For (2S,6S)-II and its
enantiom er (2R,6R)- II, th e carboxylatom ethyl and hydroxym ethyl are cis;
both occupy equatorial positions. The carboxylatom ethyl alw ays occupies an
equatorial position for all four stereoisomers.
* The num bering system for th e ORTEP draw ings of the crystal structures is
different from IUPAC nom enclature.
40
C10A
03A
C7A
C9 A
C2A
C11A
C3A
C6 A
CIA
C4 A
N1A
C5 A
C10B
C8B
04B
C7B
sly
C2B
C3B
CBB
C1B
C4B
—0
NIB
CSB
F igure II.2. ORTEP draw ing of (2S,£R)-2-hydroxymethyl-6m ethoxycarbonylm ethyl-2,4,4-trim ethylm orpholinium iodide
F igure II.3. ORTEP draw ing of (2R,6“R)-2-hydroxym ethyl-6m ethoxycarbonylm ethyl-2,4,4-trim ethylm orpholinium iodide
42
F igure II.4. ORTEP draw ing of (2R,6>£>)-6-carboxylatomethyl-2hydroxym ethyl-2,4,4-trim ethylm orpholinium
43
0 1
W
02
CIO
F igure II.5. ORTEP draw ing of (2f?,fiR)-6-carboxylatomethyl-2hydroxym ethyl-2,4,4-trim ethylm orpholinium
44
II.2.2. S yn th eses o f m eth yl (2 S ,6S,2R ,6R )- and (2S ,6 R # R ,6 S )- 2-[6-(2cyanoethyl)-4,6-dim ethylm orpholinyl]acetate, 8
11.2.2.1. P reparation o f (i?S)-5-(Ar,Ar-dim ethylam ino)-4-hydroxy-4m eth ylp en tan en itrile, (RS)-IO
P rep aratio n
of
(/?«S)-5-(A^,iV-dimethylamino)-4-hydroxy-4-methyl-
pen tan en itrile, 10, is shown in Scheme 11.11.
H N M e,
/-P rO H / H 9 0
M eO H
10
11
Schem e 11.11
F irst, 3-m ethyl-3-buten-l-ol,
12, was converted into 3-methyl-3-
butenyl m ethanesulfonate, 16, by tre atm e n t w ith m ethanesulfonyl chloride
in dichlorom ethane a t 0 °C in 98% yield. T hen tre a tm e n t of 16 w ith sodium
cyanide in
DMSO gave 4-m ethyl-4-pentenenitrile,
17, in
77% yield.
Epoxidation 8 6 of 17 w ith m onoperoxyphthalic acid (MMPP), m agnesium salt
hexahydrate, gave (f?S )-3-(l-m ethyloxiranyl)propanenitrile, 11, in 84% yield.
Compound
1 1
could also be obtained by epoxidation of 17 w ith m-
chloroperoxybenzoic acid (ra-CPBA ) . 8 7
45
By com parison w ith m-CPBA, M MPP is non-shock-sensitive and non­
deflagrating. Consequently, M MPP is m uch safer to use in both small- and
large-scale operations. In addition, both MMPP and th e oxidation byproduct,
m agnesium p h th alate, are w ater-soluble th u s easily rem oved by extraction
after completion of th e reaction.
(RS)-5-(N,ALD im ethylam ino)-4-hydroxy-4-methylpentanenitrile,
1 0
,
w as obtained by rin g opening of (R S )-ll w ith dim ethylam ine in m ethanol a t
room tem p eratu re in 90% yield. Com pound 17 was identified by com parison
of *H NMR and IR w ith lite ra tu re . 8 8
com parison of
Compound 11 was identified by
NMR w ith lite ra tu re . 8 ^ ’8 9 Compound 10 w as characterized
by *H NMR, 13C NMR, FT-IR, m ass spectroscopy, and elem ental analysis.
11.2.2.2. O ptical r eso lu tio n o f CRS)-5-dV,A/-dimethylainino)-4-hydroxy4-m eth ylp en tanenitrile, (R S )-10
(RjS)-5-(iV,N-Dimethylamino)-4-hydroxy-4-methylpentanenitrile,
10,
w as resolved as th e 3,5-dinitrobenzoate esters 18 by HPLC using a chiral
column (Scheme 11.12).
F irst, compound
10 w as tre ated w ith 3,5-
dinitrobenzoyl chloride in dichlorom ethane in the presence of triethylam ine
a t 0 °C to give a racem ic m ixture of 18 in 100% yield. T hen th is racem ic
m ixture was resolved by chiral column HPLC (CHIRALCEL OD column, 25
cm x 2 cm, J. T. B aker Inc., hexanes:ethanol = 6 : 1 ) to give two enantiom ers
(R)- an d (S)-18.
46
D N B - Cl
E t3N / C H 2C l2
OH
ODNB
,N ,
„N,
CN
HNM^
10
ODNB
18
OH
H N M e2
>
--------- s CN
chiral
colum n
CN
M eO H
M eO H
(/?)-!8
(/?)-10
HPLC
^
ODNB
H N M e2
--------- ►
*
CN
I
\
0H
M eO H
(N)-10
(S)-18
Schem e 11.12
A fter th e optical resolution of 18, compounds (R)-10 and (<S)-10 could
be obtained by am inolysis of CR)-18 an d (S)-18 respectively. We tried the
am inolysis reaction w ith (RS)-18.
D im ethylam ine (2 equivalents) was
bubbled into a solution of (R S )-18 in m ethanol. The reaction was completed
w ithin h a lf an hour, giving (i?S)-10 in 76% yield. (If we use GR)-18 or (S)-18,
we shall get CR) - 1 0 or (<S)-1 0 .)
We tried other m ethods to resolve compound 10, such as (a) m aking
q u a te rn a ry am m onium salts w ith chiral acids (tartaric acid, dibenzoyl
ta rta ric acid, 3-bromocamphor-9-sulfonic acid, W -acetyl-L-glutamic acid,
m andelic acid, an d m alic acid) followed by recrystallization and (b)
converting it into an ester w ith a chiral acid chloride ((2 S)-(+)-camphanic
chloride). These m ethods did not work.
47
II.2.2.3. P reparation o f (R)- and (S)-5-(iV,iV-dimethylamino)-4hydroxy-4-m ethylpentanenitrile, (R)- and (S)-10
A nother route for prep aration of (R )- and (S)-5-(Af,A-dimethylamino)4-hydroxy-4-m ethylpentanenitrile, 10, is shown in Scheme 11.13.
i>O
Oi.
/
✓OH
0
Swern
oxidn
( E t 0 ) 2 P (0 )C H 2CN
M eO L i
*
M eO H / C H 2C12
^
(S)-I9
(/?)-5
OH
H N M e,
Oi
"C N
M eO H
(/?)-20
H, ,Pd/C
,N.
CN
EtO H
(.R)-H)
(/<)-21
Schem e 11.13
(72)-2-Methylglycidol, 5, was converted into CR)-3 -(l-m ethyloxiranyl)- 2 propenenitrile, 20, by Sw ern oxidation of 5 followed by W ittig reaction w ith
The Sw ern oxidation reaction of 5 was
diethyl cyanom ethylphosphonate.
conducted in dichlorom ethane a t -50 to -60 °C (acetonitrile/acetone/dry ice
bath) an d 1.5 equivalents of oxalyl chloride and 3 equivalents of DMSO were
needed
for th e
completion
m ethyloxiranyl)form aldehyde,
dichlorom ethane.
dichlorom ethane.
reaction.
Compound
of the
19,
reaction.
was
After workup,
obtained
as
a
(S)-(l-
solution
19 was too volatile to separate
in
from
Therefore, th e solution was used directly for the W ittig
Diethyl cyanom ethylphosphonate was tre ated w ith lithium
m ethoxide in m ethanol to generate phosphonate carbanion.
solution of 19 in dichlorom ethane was added.
Then, the
After stirrin g overnight at
48
room tem p eratu re, 20 w as isolated in 43% yield. Compounds CR) - 2 0 an d (S)20 were prepared from CR)-5 an d (S )-5 respectively and characterized by !H
NMR and FT-IR.
Compounds (R)- and (S)-20 were tre ated w ith dim ethylam ine in
m ethanol to give (R )- an d (S)-5-(A,N-dimethylamino)-4-hydroxy-4-methyl-2pen tenenitrile, 21, respectively in 89% yield, which were characterized by
NMR an d FT-IR. C atalytic hydrogenation (10% Pd on charcoal) of (R)- and
(SO-21 in absolute ethanol a t 25 psi an d room tem p eratu re for 24 hours gave
(R)- an d (<S)-5-(N,N-dimethylamino)-4-hydroxy-4-methylpentanenitrile, 10,
respectively in 87% yield.
By th is route, compounds (R)- and (S)-10 can be m ade from (R)- and
(S)-5 respectively. But, as discussed a t th e beginning of section II. 2 . 1 , both
(R)- an d (S)-5 are not optically pure. A fter optical enrichm ent by m aking the
4-nitrobenzoate ester, 13, followed by recrystallization, how to regenerate
compound 5 is still a challenge.
11.2.2.4. A ttem pted preparation o f (R )- and (<S)-3-(l-methyloxiranyl)
propan en itrile, (R)- and (S ) -ll
We tried to p rep are (R)- and (S')-3-(l-m ethyloxiranyl)propanenitrile,
11, from (R)- and (S)-5. As show n in Scheme 11.14, compounds (R)- and (S)-5
were
converted
into
(S)-
an d
(R )-(1 -m ethyloxiranyl )methyl
4-
nitrobenzenesulfonate, 22, by reaction w ith 4-nitrobenzenesulfonyl chloride
in
dichlorom ethane
in
th e
presence
of
triethylam ine
at
0
°C.
49
o„ /
t>
o
„OH
c i s o 2^ n o 2 o
---------CH2Cl2/Et3N
L >O s02-<©>-N02h I t
t ^
c
N
-78°C
22
11
Nal
HOAc
h 2o
HO
K
34
X>SO K
^ ) ~ no2
23
S c h e m e 11.14
R ecrystallization of 22 from ethanol provided light yellow crystals. In order
to m easure th e optical p u rity of 22 by M osher's ester an aly sis , 8 2 compound
22
was
converted
into
2-hydroxy-3-iodo-2-methylpropyl
4-
nitrobenzenesulfonate, 23, by reaction w ith sodium iodide in acetic acid and
w a te r . 9 0
C rystals of 23 w ere obtained by recrystallization from m ethanol.
U nfortunately, compound 23 could not be transform ed into M osher's ester
because it is a te rtia ry alcohol. The conversion of 22 into 11 failed. K lunder
et a l 9 1 studied th e reactivities of glycidyl arenesulfonate derivatives tow ard
different organom etallic reagents.
In m ost cases studied, rin g opening
occurred. Johnson an d D u tra 9 2 reported th a t tosylates and epoxy rings had
sim ilar reactivities tow ard lith iu m diorganocuprates. Therefore, compounds
(R )- and (<S) - 1 1 could not be prepared this way.
50
Compounds 22 an d 23 w ere identified by !H NMR and verified by
single-crystal X-ray analyses.93,94
II.2.2.5. Structural an alysis o f CR)-2-hydroxy-3-iodo-2-methylpropyl
4-nitrobenzen esu lfonate, CR)-23
For compound CR)-23 (Figure 11.6),^ we used PCM ODEL 9 5 to calculate
th e eighty-one possible staggered conformations of th e four torsion-angle
sequence—IC-C-C-O-SC^Ar. For the global m inim um , those torsion angles
are -58.9°, -55.2°, 169.3°, an d -83.6°, respectively.
In th e crystal, they are
-57.7 (4)°, -61.7 (4)°, 171.2 (2)°, and -73.0 (3)°, respectively.
In this
conformation, th e I atom is on top of the benzene rin g w ith nearly equal
distances betw een th e I atom and six C atom s of th e benzene ring.
The
intram olecular distance [4.083 (2) A] betw een I and th e centroid of th e aryl
rin g is alm ost equal to th e sum (3.90 A) of th e van der W aals rad iu s of I and
th e thickness of th e benzene rin g . 9 6
W ith respect to bond distances and
angles, PCMODEL agrees approxim ately w ith the X-ray determ ination.
N otable exceptions are th e C1-C2, C7-C8, and C8-C9 bond lengths and the
C7-C8-C9 an d 0 5 -N -0 6 bond angles, as well as m any values of the SO 3
group. The X-ray resu lts reveal th a t the 02-C3 bond is 0.049
A longer th a n
th e 01-C 2 bond, which is consistent w ith OSC^Ar being a b etter leaving
group th a n th e OH group.
Molecules are linked in chains along the
crystallographic b axis by w eak interm olecular hydrogen bonds involving
F igure II.6. ORTEP draw ing of (f?)-2-hydroxy-3-iodo-2m ethylpropyl 4-nitrobenzenesulfonate
52
O q .h a n d
Os=o-
The 0 1 —0 3 distance is 2.927 (4)
A and the
O -H-O angle is
165 (4)°.
II.2.2.6. Syn th eses o f m ethyl (2S,6S;2R,6R)- and (2S,6R,2R,6S)- 2-[6-(2cyanoethyl)-4,6-dim ethylm orpholinyl] acetate, 8
M ethyl
(2S,6S;2R,6R )-
dim ethylm orpholinyljacetate,
8
and
(2S,6R;.2R,6S)-2-[6-(2-cyanoethyl)-4,6-
, were synthesized startin g from (RS)-5-(N,N-
dim ethylam ino)-4-hydroxy-4-m ethylpentanenitrile, 10, as shown in Scheme
11.15.
COOMe
COOMe
COOM e
PliSH
2. M eO H
COOMe
COOMe
colum n
S i0 2
E tO A c/M eO H
Schem e 11.15
Racemic m ixture of 10 reacted w ith
1
equivalent of m ethyl 4-bromo-2-
butenoate in nitrom ethane a t 50 to 60 °C for 3 horn's.
T hen solvent was
changed to m ethanol an d refluxed for 18 hours.
In nitrom ethane,
condensation of 10 w ith 3 took place to produce acyclic q u atern ary am ine
53
salt. In m ethanol, rin g closure reaction occurred. The ring closure reaction
cannot be carried out in THF, as in th e reaction of 14, due to th e insolubility
of th e acyclic interm ediate. C oncentration of the reaction m ixture followed
by w ashing w ith diethyl eth e r an d drying u nder vacuum gave crude product
(2iS,,^S;2JR,6/2;2S,6Z2;2i2,dS)-2-(2-cyanoethyl)-6-(methoxycarbonyl)methyl2,4,4-trim ethylm orpholinium bromide, 7, in quantitative yield. This m ixture
of four stereoisom ers could n ot be separated by norm al-phase HPLC because
they were q u a tern ary am ine salts.
We tried reversed-phase HPLC to
separate th e two p airs of racem ates, (2R,6R\2S,6S)-1 and {2S,6R\2R,6S)-1 ,
an d we failed. The *H NMR spectrum of 7 was too complicated to in terp ret
because it was a m ixture of four stereoisomers. Therefore, th e crude m ixture
of 7 w as used for next reaction w ithout fu rth e r purification, separation, and
characterization.
Crude 7 w as tre a te d w ith 3 equivalents of thiophenol in N,Ndim ethylethanolam ine 9 7 a t 70 °C to give m ethyl (2S,6S',2R,6R;2S,6R',2R,6S)2-[6-(2-c.yanoethyl)-4,6-dimethylmorpholinyl]acetate,
8
, which was separated
by column chrom atography to give two racem ic m ixtures, (2S,6S;2R,6R)-8 as
a solid an d (2S,6R]2R,6S)-8 as an oil, w ith a ratio of 3:4 and total yield of
25%. The low yield of the dem ethylation reaction m ay be caused m ainly by
th e attack of thiophenol on the ester group of 7.98>" The ratio of the two
racem ic m ixtures indicates th a t the stereoselectivity of the rin g closure
reaction is not significant.
The slight preference for (2S,6R;2i?,6S)-7 over
{2R,6R\2S,6S )-7 m ay be a ttrib u ted to chair tran sitio n structures in th e ring
54
closure reaction (Figure II.7). The 2-cyanoethyl group is bigger th a n m ethyl
group an d tends to be cis w ith (methoxycarbonyl)m ethyl so th a t both
cyanoethyl an d (methoxycarbonyl)methyl can adopt equatorial positions.
C rystals
of
(2S,6S;2R,6R ) - 8
recrystallization
from
for
X-ray
hexanes.
(2S,6R;2R,6S)-8 were characterized by
analysis
Compounds
were
obtained
(2S,6S;2R,6R)-S
by
and
NMR, 13C NMR, FT-IR, m ass
spectroscopy, and elem ental analyses.
CN
Me
+
1'
F igu re II.7. T ransition stru ctu res for ring closure reaction of 10
The stru ctu re of (2S,6S]2R,6R ) - 8 was verified by single-crystal X-ray
analysis (Figure II. 8 ) . l "
The m orpholine rin g is in a chair conformation.
The m ethoxycarbonylm ethyl an d m ethyl on C3 are cis; both occupy
equatorial positions, as does th e IV-methyl group. We used PCMODEL 9 5 to
calculate th e eighty-one possible staggered conformations of the four torsionangle sequence—NC-CH 2 -CH 2 -C-0 an d 0-C H -C H 2 -C (0)-0C H 3.
For the
global m inim um , which was also found by GMMX , 1 0 1 those torsion angles
are 179.2°, -50.5°, -60.0°, an d 140.2°, respectively. In the crystal, they are
55
C12
C ll
CIO
F igure II.8. ORTEP draw ing of (^.S,)dS;2R,dR)-2-[6-(2-cyanoethyl)-4>6dim ethylm orpholinyl]acetate
56
-170.0 (1)°, -45.9 (2 )°, -71.6 (2)°, an d 142.8 (1)°, respectively. W ith respect to
bond distances
an d
angles,
PCMODEL also agrees
w ith
th e
X-ray
determ ination.
(R )- or (S)-5-(N,N-
In th is synthetic route, if we s ta rt w ith
dim ethylam ino)-4-hydroxy-4-m ethylpentanenitrile,
10,
which
can
be
obtained by optical resolution, in place of CRS)-1 0 , we shall be able to get the
four stereoisom ers of 8 sep arated (Scheme 11.16).
'CN
(S) - 1 0
(7?)-10
NN
✓✓
COOM e
\
\
COOMe
(2S, 67?)-8
COOMe
(27?, 67?)-8
(.2S , 6 .S')-8
N
COOMe
(27?, 6S )-8
Schem e 11.16
11.2.3. Sum m ary
(.2R,6R )-,
(2S,6S )-,
(2R,6S )-,
and
(2 S , 6 i?)-6 -carboxylatom ethyl- 2 -
hydroxjnnethyl-2,4,4-trim ethylm orpholinim n, ta rg e t molecule II, and m ethyl
57
(2S,6S]2R,6R )-
an d
m orpholinyl]acetate
8
C2S,6R;2.R,6S)-2-[6-(2-cyanoethyl)-4,6-dimethylwere synthesized.
(i?S)-5-(A^A^-Dimethylamino)-4-
hydroxy-4-m ethylpentanenitrile, 10, w as resolved.
The m ethod for synthesizing ta rg e t molecule II in section II.2.1 can be
fu rth e r extended for synthesizing a series of other inhibitors by changing
hydroxy group of 2, 1 or ta rg e t molecule II to other groups, which will be
discussed in C h ap ter IV. T arget molecule III can also be approached by this
strateg y so th a t th e u ltim ate goal of th is project can be achieved.
By synthetic route in section II.2.2, we got compound
8
, from which
ta rg e t molecule III can be derived. B ut by this route we cannot get other
inhibitors which can be obtained by the extension of the m ethod for
synthesizing ta rg e t molecule II (see C hapter IV).
II.3.
EXPERIMENTAL
II.3.1. G eneral procedures
U ncorrected m elting points were m easured on an Electrotherm al
m elting point ap p aratu s,
^H NMR spectra were recorded w ith a B ruker
AMX 500, AM 400, AC 200, or AC 100 FT-NMR spectrom eter a t 500, 400,
200, or 100 MHz, respectively.
13C NMR spectra were recorded w ith a
B ru k er AC 200 FT-NMR spectrom eter a t 50 MHz. U nless noted otherwise,
all NMR spectra were recorded in CDCI3 .
expressed
in
p a rts
per
m illion
(ppm)
Proton chemical shifts are
down
field
from
in tern al
tetram eth y lsilan e (TMS); coupling constants th a t were verified using PANIC
(P aram eter A djustm ent in NMR by Iteration Calculation) are listed as J ;
observed coupling constants not verified are listed as J app', all coupling
constants are reported in Hz. 13C chemical shifts are also expressed in ppm
relative to th e solvent chemical shift. A ssignm ents of the *H and 13C NMR
signals were m ade by com parison w ith sim ilar known compounds and using
DEPT, 2D
13
C-1H correlation, and 2D *H COSY experim ents.
Infrared
spectra were recorded on a P erk in E lm er 1760X FT-IR spectrom eter as th in
films on KBr cells and are reported in cm-1. M ass spectra were obtained w ith
a H ew lett-Packard 5971A GC-MS.
FAB MS sam ples were prepared by
suspending in glycerol an d th e spectra were recorded on a F innigan TSQ 70.
Elem ental analyses were perform ed by O neida R esearch Services of
W hitesboro, New York. The optical rotations were recorded in a 3.5 x
1 0
mm
or 10 x 100 m m cell w ith a JASCO DIP-370 digital polarim eter.
U nless otherwise noted, m aterials were obtained from commercial
sources an d used w ithout fu rth e r purification. T etrahydrofuran (THF) was
distilled from potassium . D iethyl eth er was distilled from sodium -potassium
alloy. Dim ethyl sulfoxide (DMSO) was distilled from CaH 2 u n d er reduced
pressu re and stored over Linde M olecular Sieves Type 3A in a sealed brown
bottle.
T riethylam ine w as distilled from CaH 2 and stored over Linde
M olecular Sieves Type 3A. Dichlorom ethane was purified by shaking w ith
concd H 2 SC>4 , w ashing w ith H 20
and satd brine, drying w ith CaH 2,
distilling, an d storing over 4A M olecular Sieves. M ethanol was distilled over
a sm all am ount of Mg. 3,5-Dinitrobenzoyl chloride, 4-nitrobenzoyl chloride,
59
and 4-m trobenzenesulfonyl chloride were recrystallized from hexanes.
Thionyl chloride was distilled before use. M ethyl 4-brom o-2-butenoate was
purified by vacuum distillation.
The organic solutions were dried (M gSO ^ and concentrated by rotary
evaporation unless otherw ise noted.
II.3.2. P roced ures
(/?)-(l-M ethyloxiranyl)m ethyl
4-nitrobenzoate
(CR)-13).
A
solution of 4-nitrobenzoyl chloride (116 mmol, 21.6 g) in CH 2 CI2 (30 mL) was
slowly added to a stirred m ixture of (S')-2-methylglycidol (110 mmol, 9.98 g,
97% purity) an d EtgN (124 mmol, 12.6 g) in CH 2 Cl 2 (90 mL) a t 0 °C (icew ater bath). The reaction m ixture was stirred for additional
2
h at
°C. The
0
reaction m ixture was diluted w ith cold CH 2 CI2 (60 mL) and filtered w ith
suction to remove precipitated am m onium salt, which th en was w ashed w ith
cold CH 2 CI2 (3 x 15 mL). The m other liquor w as w ashed w ith cold 5% HC1
(60 mL), cold H 20 (2 x 40 mL), an d dried. C oncentration of th e solution gave
25.54 g of a light yellow solid. R ecrystallization from EtO H (300 mL) gave
19.81 g of light yellow crystals. The crystals were dissolved in E t20 (250 mL)
while refluxing and th e n th e solution was cooled slowly to
1 0
°C, giving 15.65
g of crystals, which were th en recrystallized from (i-Pr)20 (130 mL) to give
12.8 g (49 %, ee > 97%) of (R)-13 (mp 87.5-89.0 °C, L it . 8 3 85.5-86.5 °C).
1H NMR (200 MHz; Lit.83): 1.49 (s, 3 H, CH z-), 2.76 (d, 1 H, tf-C H =oxirane,
J app = 4.6), 2.87 (d,
1
H, H-CH=oxirane, J app = 4.5), 4.24 (d,
1
H, H-
60
CHOCOAr, J app = 11.9), 4.58 (d, 1 H, tf-CHOCOAr, J app = 12.0), 8.21-8.34
(m, 4 H, Bz). [a]23D -5.85° (c 2.035, CHC13, 10 x 100 m m cell. L it . 3 3 -5.87°).
(S)-13 was prepared from 0R)-2-methylglycidol the sam e way as CR)-13. mp
87.5-89.0 °C. [a]23D +5.83° (c 1.989, CHC13 10 x 100 m m cell).
(7?)-3-M ethylam iiio-2-m ethylpropane-l,2-diol (CR)-4). A solution
of Cff)-13 (100 mmol, 23.7 g) in MeOH (200 mL) was added dropwise to a
solution of H 2 NMe (0.6 mol, 18.6 g) in MeOH (60 mL).
The m ixture was
stirred for 3 h. The precipitate was removed by filtration. The solution was
placed in a refrigerator (0 °C) for 0.5 h th e n in a freezer for
1
h. Again, the
precipitate was removed by filtration. The solution was concentrated u n til a
large am ount of precipitate appeared and th en placed in a freezer for 0.5 h.
Again, th e precipitate w as removed by filtration.
concentrated and th en placed u nder vacuum .
The solution was
The residue was distilled
u n d er vacuum affording 8.92 g (75%) of CR)-4 as a colorless oil ( bp 80 °C/0.2
Torr, 160 °C/17 Torr).
1H NMR (200 MHz): 1.10 (s, 3 H, CH 3 -C), 2.44 (s, 3 H, CH 3 -N), 2.66 (d, 1 H,
H-CHN, J app = 12.1), 2.74 (dd, 1 H, H-CHN, J app = 12.2, 1.4), 3.48 (dd, 1 H,
H-CHOH, J app = 11.2, 1.4), 3.65 (d, 1 H, H-CHOH, J app = 11.0). 13C NMR:
23.2 (CH 3 -C), 36.9 (CH 3 -N), 60.9 (-CH 2 -N), 70.9 (C-OH), 71.2 (-CH 2 OH). IR:
3321 (OH), 1055 (C-N). MS m /e (relative intensity):
8 8
(11.4), 75 (3.6), 70
(7.3), 58 (10.4), 57 (4.3), 45 (5.1), 44 (100), 43 ( 1 0 .2 ), 42 ( 1 1 . 1 ). Anal. Calcd
for C 5 H 1 3 N 0 2: C, 50.42; H, 10.92; N, 11.76. Found: C, 50.14; H, 11.02; N,
11.57.
61
(iS')-4 was p rep ared from CR)-13 the same w ay as CR)-4.
Anal. Found: C,
50.29; H, 10.89; N, 11.56.
(R)-M ethyl
4-[m ethyl-(2,3-dihydroxy-2-m ethylpropyl)am ino]-2-
bu ten oate (CR)-14). A solution of m ethyl 4-brom o-2-butenoate (40.34 mmol,
7.22 g) in TH F (30 mL) w as added to a m ixture of (R)-4 (40.34 mmol, 4.80 g)
and K 2 CO 3 (54 mmol, 7.4 g) in THF (50 mL).
The reaction m ixture was
stirred overnight an d th e n filtered. The solution was concentrated and then
placed u n d er vacuum . D ry E t 2
0
(40 mL) was added an d the precipitate was
removed by filtration. C oncentration of the solution gave 7.26 g (83%) of a
light yellow oil, which w as used for next reaction w ithout fu rth er
purification.
!H NMR (200 MHz): 1.07 (s, 3 H, CH 3 -C), 2.38 (s, 3 H, CH 3 -N), 2.49 (d, 1 H,
H-CHC(OH)(CH 3 )CH 2 OH,
J app
=
13.8),
2.62
(d,
1
H,
H-
CHC(OH)(CH 3 )CH 2 OH, J app = 13.8), 3.13-3.63 (m, 4 H, H 2 C-CH=CH-, H 2COH), 3.73 (s, 3 H, CH 3 -O), 5.90-6.00 (m, 1 H, H C -C 0 2 CH3), 6.85-6.99 (m,
1
H, H C =C H -C 0 2 CH 3). 13C NMR (50 MHz): 23.7 (CH 3 -C), 45.0 (CH 3 -N), 51.6
(CH 3 O), 60.4 (CH 2 -CH=CH), 65.4 (N-CH 2 -COH), 70.2 (CH 2 -OH), 71.5 (COH), 123.2 (=C H -C 0 2 CH3), 144.9 (CH =CH C0 2 CH3), 166.4 (C 0 2 CH3).
IR:
3425 (OH), 1724 (C=0), 1660 (C=C). MS, FAB, m / e : 218 (M++1).
(iS)-14 was p rep ared from (S )-17 th e sam e w ay as (i?)-14.
M ethyl (2S,6'i?)-2-(4,6-dim ethyl-6-hydroxym ethylm orpholinyl)
a ceta te
((2S,6R)-2) and m eth yl (2i2,fiR)-2-(4,6-dimethyl-6-hydroxy-
m ethylm orpholinyl) a ceta te ((2R,6R)-2). DBU (2.5 mL, 16.4 mmol) was
62
added to a solution of crude CR)-14 (33.4 mmol, 7.26 g) in TH F (500 mL). The
reaction m ixture w as stirred a t r t for 24 h.
C oncentration of the solution
gave a brown liquid, which was purified by column chrom atography (S i0 2,
70-230 m esh, sam ple:Si 0
2
of two diastereom ers.
chrom atography
(S i0 2,
= 1:25, EtOAc:MeOH = 100:5), yielding a m ixture
The diastereom ers were separated by column
230-400
mesh,
sam ple:Si 0
2
=
1:70,
hexanes:C H 2 Cl2:EtOH = 100:100:30), giving (2S,6R )-2 (2.18 g, 30%) and
(2R,6R )-2 (0.70 g, 9.6%).
(2S,6R)- 2:
1H NMR (500 MHz): 1.11 (s, 3 H, CH 3 -C 6 ), 1.74 (dd, 1 H, H(ax)-C3, J =
10.87, 10.73), 1.94 (d, 1 H, H(ax)-C5, J = 11.83), 2.20 (s, 3 H, CH 3 -N), 2.37
(dd, 1 H, # -C H C 0 2 CH3, J = 16.35, 4.06), 2.49 (dd, 1 H, H -C H C 0 2 CH3, J =
16.35, 8.93), 2.65 (d, 1 H, H(e q)-C5, J = 11.83), 2.70 (d, 1 H, H(e q)-C3, J =
10.87), 3.40 (d, 1 H, H-CHOH, J = 11.58), 3.70 (s, 3 H, CH 3 -0), 4.20 (d, 1 H,
H-CHOH, J = 11.58), 4.29-4.37 (m, H- C2).
13C NMR: 23.6 (CH 3 -C), 38.2
(CH 2 -C 0 2 CH3), 45.9 (CH 3 -N), 51.7 (CH 3 -0), 58.8 (C3), 61.3 (C5), 65.8 (CH2OH),
6 6 .6
(C2), 73.5 (C 6 ), 171.5 (C=0). IR: 3506 (OH), 1741 (C=0). MS m /e
(relative intensity): 217 (19.5), 186 (54.5), 144 (13.7), 143 (26.5), 142 (31.6),
128 (17.6), 114 (10.5), 98 (18.4), 70 (25.1), 59 (16.9), 58 (17.6), 57 (21.5), 44
(47.9), 43 (100), 42 (60.4), 41 (27.0). [a]23D, -20.2° (c 9.95, CHC13, 3.5 x 10
mm cell). Anal. Calcd for C 1 0 H 1 9 NO 4 : C, 55.30; H, 8.76; N, 6.45. Found: C,
54.98; H, 8.73; N, 6.47.
(2R,6R)-2:
*H NMR (500 MHz): 1.29 (s, 3 H, C tf 3 -C 6 ), 1.68 (dd, 1 H, H(ax)-C3, J =
11.08, 10.96), 2.09 (d, 1 H, H( ax)-C5, J = 10.95), 2.24 (s, 3 H, CH 3 -N), 2.39
(dd, 1 H, H -C H C 0 2 CH3, J =15.30, 5.74), 2.42 (d, 1 H, #(eq)-C 5, J = 10.95),
2.48 (dd,
1
H, H-CHCO 2 CH 3 , J = 15.30, 7.11), 2.75 (d, 1 H, H(eq)-C3, J =
11.08), 3.33 (d,
1
H, tf-CH O H , J = 11.23), 3.47 (d,
3.69 (s, 3 H, CH 3 -O), 4.21-4.27 (m, 1 H, H- C2).
1
H, tf-CH O H , J = 11.23),
NMR: 18.7 (CH 3 -C), 38.9
(CH 2 -CO 2 CH 3 ), 46.4 (CH 3 -N), 51.6 (CH 3 -O), 59.3 (C3), 59.7 (C5), 66.5 (C2),
69.2 (CH 2 -OH), 74.3 (C 6 ), 171.1 (C=0). IR: 3452 (OH), 1741 (C=0). MS m /e
(relative intensity): 217 (21.1), 186 (56.2), 144 (16.1), 143 (26.5), 142 (37.4),
128 (15.0), 114 (13.3), 98 (17.7), 84 (84.8), 70 (26.0), 59 (15.6), 58 (12.7), 57
(19.2), 44 (48.3), 43 (100), 42 (40.7), 41 (15.3). [a]23D, +2.24° (c
1 1
.6 , CHCI 3 ,
3.5 x 10 m in cell). Anal. Calcd for C 1 0 H 1 9 NO 4 : C, 55.30; H, 8.76; N, 6.45.
Found: C, 54.90; H, 8.45; N, 6.33.
(2R,6S)- 2, (2S,6S)-2 were prepared from (S)-14 the sam e w ay as (2S,6R )-2
and (2R,6R)- 2.
(2R,6S)- 2:
[oc]23d +20.6° (c 5.93, CHCI3 , 3.5 x 10 m m cell). Anal. Found: C, 54.98; H,
8.45; N, 6.46.
(2S,6S)- 2 :
[a]23£> -2.26° (c 9.28, CHCI 3 , 3.5 x 10 mm cell) . Anal. Found: C, 54.92; H,
8.50; N, 6.34.
(2/?,67S)-2-Hydroxymethyl-6-methoxycarhonylmethyl-2,4,4-trim ethylm orpholinium iod id e (C2R,ftS)-l).
To a solution of (2S,6R)-2
64
(0.868 g, 4 mmol) in dry E t 2
0
(30 mL) w as added CH 3 I (5 mL, 80 mmol). The
m ixture was placed in th e d ark and stirred for 3 d.
decanted an d th e precipitate w as w ashed w ith dry E t 2
The solution was
0
( 3 x 5 mL). The
yellow p aste w as dried u n d er vacuum to give 1.06 g (74%) of a light yellow
solid which was used for th e next reaction w ithout fu rth e r purification.
C rystals for X-ray analysis w ere obtained by recrystallization from MeOH by
vapor diffusion w ith E t 2
0
.
(2R,6S)-1 (mp 136.5-140 °C, dec.):
!H NMR (400 MHz, CD 3 OD, TMS): 1.27 (s, 3 H, CH 3 -C2), 2.60 (dd, 1 H, HC H C 0 2 CH3, J = 16.13, 7.08), 2.65 (dd, 1 H, H -C H C 0 2 CH3, J = 16.13, 5.04),
3.13 (d, 1 H, H{ ax)-C3, J = 13.39), 3.26 (dd, 1 H, H( ax)-C5, J = 11.91, 12.82),
3.30 (s, 3 H, CH 3 (eq)-N), 3.36 (s, 3 H, CH 3 (ax)-N), 3.56 (d, 1 H, H-CHOH, J =
11.69), 3.63 (ddd, 1H, #(eq)-C 5, J = 12.82, 1.56, 1.49), 3.71 (s, 3 H, CH30),
3.83 (dd, H(eq)-C3, J = 13.39, 1.56), 3.95 (d, 1 H, H-CHOH, J =11.69), 4.634.70 (m, 1 H, H-C6).
13C NMR (CD 3 OD): 26.94 (CH 3 -C 2 ), 38.31 (CH2-
C 0 2 CH3), 51.52 (CH 3 (ax)-N), 52.56 (CH 3 0 ), 59.53 (CH 3 (eq)-N), 63.74 (C3,
C 6 ), 63.96 (C5), 64.10 (CH 2 OH), 74.98 (C2), 171.72 (C 0 2 CH3).
IR: 3346
(OH), 1734 (C=0), 1058 (C-O-C). MS, FAB, m/e: 232 (M-L). [a]22D -21.3° (c
7.10, MeOH, 3.5 x 10 mm cell). Anal. Calcd for C n H 2 2 N 0
4
I: C, 36.77; H,
6.13; N, 3.90. Found: C, 36.64; H, 6 .0 2 ; N, 3.87.
(2R,6R)- 1, (2S,6S)-1, an d (2S,6R )-1 were prepared the sam e w ay as {2R,6S)~ 1
from (2R,6R)- 2, (2S,6S)-2, an d (2R,6S )-2 respectively.
(2R,6R )-1 (mp 136.2-140 °C, dec.):
1H NMR (400 MHz, CD 3 OD, TMS): 1.45 (s, 3 H, CH 3 -C2), 2.63 (dd, 1 H, HC H C 0 2 CH3, J = 15.38, 8.41), 2.67 (dd, 1 H, H -C H C 0 2 CH3, J =15.38, 4.91),
3.20 (dd, 1 H, H( ax)-C5, J = 12.05, 11.74), 3.31 (s, 3 H, C # 3 (eq)-N), 3.34 (d, 1
H, iT-CHOH, J = 11.71), 3.43 (s, 3 H, CH 3 (ax)-N), 3.45 (d, 1 H, tf(ax)-C3, J
=13.74), 3.46 (d, 1 H, H-CHOH, J = 11.71), 3.57 (dd, 1 H, H(eq)-C3, J = 13.74,
I.84), 3.64 (ddd, 1 H, #(eq)-C 5, J = 11.74, 2.18, 1.84), 3.71 (s, 3 H, CH 3 0),
4.61-4.68 (m, 1 H, H- C 6 ).
NMR (CD 3 OD): 19.93 (CH 3 -C2), 38.13
(CH 2 C 0 2 CH3), 51.90 (CH 3 (ax)-N), 52.49 (CH 3 0 ), 59.74 (CH 3 (eq)-N), 63.21
(C 6 ), 64.34 (C5), 65.39 (C3), 69.92 (CH 2 OH), 74.77 (C2), 171.62 (C 0 2 CH3).
IR: 3359 (OH), 1735 (C=0), 1066 (C-O-C). MS, FAB, m / e : 232 (M-P). [a]22D
+15.1° (c 9.90, MeOH, 3.5 x 10 m m cell). Anal. Calcd for C n H 2 2 N 0
4
l: C,
36.77; H, 6.13; N, 3.90. Found: C, 36.67; H, 6.08; N, 3.85.
(2S,6S)- 1:
[ a ] 2 2 D
-14.8° (c 9.25, MeOH, 3.5 x 10 mm cell). Anal. Found: C, 36.76; H,
6.08; N, 3.84.
(2S,6R)- 1:
[ a ] 2 2 D
+21.5° (c 6.40, MeOH, 3.5 x 10 m m cell). Anal. Found: C, 36.66; H,
6.05; N, 3.84.
(2R,6VS)-6-Carboxylatomethyl-2-hydroxyinethyl-2,4,4-trimethyl
m orpholinium (C2R,ftS)-II). A solution of (2R,6S)-1 (0.95 g, ~ 2.6 mmol) in
0.1 M N aO H (26 mL, 2.6 mmol) was stirred a t r t overnight. The reaction
m ixture was concentrated by rotary evaporation, th en dried u n d er vacuum.
The residual solid was dissolved in CH 3 OH (30 mL) and filtered. The liquid
66
w as concentrated by ro tary evaporation an d dried u n d er vacuum .
The
resu ltin g lig h t yellow solid was dissolved in a m inim um am ount of CH 3 OH,
an d acetone (~ 40 mL) w as added.
The solution was decanted and the
precipitate was dried u n d er vacuum .
The solid obtained w as dissolved in
CH 3 OH (80 mL). T hen acetone (900 mL) w as added. The solution w as left
open to th e a ir on th e bench, yielding 0.44 g (67%) of colorless crystals
(C 1 0 H 1 9 NO 4 -2H 2 O).
(2R,6S)-ll (mp 230-231 °C):
XH NMR (400 MHz, CD 3 OD, TMS): 1.26 (s, 3 H, CH 3 -C2), 2.30 (dd, 1 H, HC H C 0 2 CH3, J = 15.34, 6.44), 2.46 (dd, 1 H, H -C H C 0 2 CH3, J = 15.34, 6.61),
3.06 (d, 1 H, H( ax)-C3, J = 13.40), 3.11 (dd, 1 H, #(ax)-C 5, J = 12.55, 11.79),
3.23 (s, 3 H, GfiT3 (eq)-N), 3.30 (s, 3 H, C # 3 (ax)-N), 3.56 (ddd, 1 H, H(eq)-C5, J
= 12.55, 1.79, 1.68), 3.64 (d,
1
H, tf-C H O H , J = 11.80), 3.75 (dd, 1 H, H(eq)-
C3, J = 13.40, 1.79), 3.87 (d,
1
H, H-CHOH, J = 11.80), 4.49-4.57 (m, 1 H, H-
C 6 ).
13C NMR ( CD 3 OD): 26.84 (CH 3 -C2), 42.21 (CH 2 C 0 2 CH3), 51.24
(CH 3 (eq)-N), 59.25 (CH 3 (ax)-N), 63.89 (CH 2 OH, C5), 64.92 (C 6 ), 64.99 (C3),
74.65 (C2 ), 176.94 (C 0 2‘). IR: 3387 (OH), 1586 (C=0), 1061 (C-O-C). MS,
FAB, m /e: 218 (M++1). [a]22D -23.1° (c 6.50, MeOH, 3.5 x 10 m m cell). Anal.
Calcd for C i 0 H 1 9 NO 4 -2H 2 O: C, 47.43; H, 9.09; N, 5.53. Found: C, 47.78; H,
8.93; N, 5.46.
C2i?,6R)-II, (2S,6S)-1I, an d (2S,6“i?)-II were p repared th e sam e way as
(2R,6S )-II from (2R,6R)-1, (2S,6S)- 1, (2S,6R )-1 respectively.
(2R,6R )-II (mp 235.5-236.5 °C):
!H NMR (400 MHz, CD 3 OD, TMS): 1.42 (s, 3 H, CH 3 -C2), 2.34 (dd,
1
H, H-
C H C 0 2 CH3, J = 14.82, 6.43), 2.45 (dd, 1 H, H -C H C 0 2 CH3, J = 14.82, 6.26),
3.09 (dd, 1 H, H{ ax)-C5, J = 12.26, 11.26),3.24 (s, 3 H, CH 3 (eq)-N), 3.30 (d,
1
H, H{ ax)-C3, J = 11.90), 3.35 (s, 3 H, CH 3 (ax)-N), 3.42 (s, 2 H, CH 2 OH), 3.46
(d, 1 H, #(eq)-C 3, J = 11.90), 3.57 (d,
H- C 6 ).
1
H, H(eq)-C5, J = 12.26), 4.51 (m, 1 H,
13C NMR (CD 3 OD): 20.00 (CH 3 -C2), 42.36 (CH 2 C 0 2 CH3), 51.65
(CH 3 (eq)-N), 59.53 (CH3(ax)-N), 64.48 (C6 ), 65.23 (C5), 65.43 (C3), 70.06
(CH 2 OH), 74.44 (C 2 ), 177.09 (C 0 2-). IR: 3583 (OH), 1587 (C=0), 1078 (C-OC). MS, FAB, m / e : 218 (M++ 1 ). [a]22D +15.6° (c 4.8, MeOH, 3.5 x
1 0
mm
cell). Anal. Calcd for C ig E I ig N O ^ H ^ : C, 47.43; H, 9.09; N, 5.53. Found: C,
47.31; H, 8.83; N, 5.47.
(2S,6S)- II:
[a]22D -16.0° (c 5.00, MeOH, 3.5 x
1 0
m m cell). Anal. Found: C, 47.29; H,
8.77; N, 5.57.
(2S,6R)- II:
[oe]22D +23.1° (c 5.40, MeOH, 3.5 x 10 m m cell). Anal. Found: C, 47.05; H,
8.89; N, 5.34.
3 -M e th y l-3 -b u te n y l
m e th a n e s u lf o n a te
(16).
A
solution
of
m ethanesulfonyl chloride (0.42 mol, 49.0 g) dissolved in CH 2 C12 (100 mL)
w as added dropwise to a stirred solution of 3-m ethyl-3-buten-l-ol (0.40 mol,
35.50 g) an d E t3N (0.60 mol, 60.6 g) in CH 2 C12 (400 mL) a t
0
°C (ice-water
bath). A fter completion of th e addition, th e reaction m ixture was stirred for 2
h a t 0 °C. The reaction m ixture was diluted w ith cold CH 2 C12 (200 mL) and
68
filtered w ith suction to remove precipitated am m onium sa lt which was th en
w ashed w ith cold CH 2 CI2 (100 mL). The m other liquor was w ashed w ith cold
5% HC1 (200 mL), cold H 2 O
(2
x 150 mL), and dried. C oncentration of the
solution gave 64.29 g (98%) of a brown oil, which was used w ithout fu rth er
purification.
1H NMR (200 MHz; Lit. 102); 1.78 (s, 3 H, C tf 3 -C=C), 2.47 (t,
Japp = 6 .8 ), 3.01 (s, 3 H, C tf 3 -S), 4.33 (t,
2
2
H, -C # 2 -C=C,
H, -CH 2 -OS, J app = 6.9), 4.84 (m,
2
H, CH2=C).
4-M ethyl-4-pentenenitrile (17). U nder N 2 , a solution of 16 (0.39
mol, 64.29 g) in DMSO (100 mL) was added dropwise to a stirred slurry of
N aCN (0.60 mol, 31.9 g) in DMSO (250 mL) a t 80 °C. After th e completion of
th e addition, th e reaction m ixture was stirred for 5 h a t 80 °C, cooled, poured
into i c e - ^ O (800 mL), an d extracted w ith E t 2
0
(4 x 800 mL). The combined
extracts were w ashed w ith satd brine (3 x 800 mL) and dried. C oncentration
of th e soluton gave a yellow oil. Vacuum distillation afforded 28.68 g (77%)
of a colorless oil (bp 65-66 °C/17 Torr; L it . 8 8 50-52 °C/12 Torr).
!H NMR (200 MHz; Lit.88): 1.77 (s, 3 H, C ff3-), 2.30-2.55 (m, 4 H,
CH2CH2C m , 4.80-4.88 (m,
2
H, CH2=C). 13C NMR: 15.5 (C2), 21.7 (CH3),
32.7 (C3), 112.0 (C5), 119.1 (CN), 141.4 (C4). IR (Lit.88): 3081 (olefinic C-H
stretch), 2247 (C=N), 1653 (C=C stretch), 898 (olefinic C-H bend).
C R S)-3-(l-M ethyloxiranyl)propanenitrile
(11).
A solution
of
m onoperoxyphthalic acid, m agnesium salt hexahydrate (70 mmol, 43.30 g,
80% purity) in H 2 O (200 mL) w as added dropwise to a solution of 17 (100
69
mmol, 9.50 g) in i-PrOH (60 mL). The m ixture was stirred for 5 h a t rt. After
addition of H 2 O (100 mL), th e reaction m ixture was extracted w ith CH 2 CI2 (3
x 150 mL). The combined extracts were w ashed w ith 1 M N a 2 S 0
10% N a 2 CC> 3 (100 mL), H 2 O
(2
3
(100 mL),
x 200 mL), dried, and concentrated affording
a colorless oil. Vacuum distillation yielded 9.32 g (84%) of a colorless oil (bp
83-84 °C/17 Torr; L it . 8 9 110 °C/25 Torr).
XH NMR (200 MHz, L it.8?): 1.36 (s, 3 H, CH 3-), 1.95 (t, 2 H, -CH 2 CH 2 CN,
J app = 7.4), 2.43 (t, 2 H, CH 2 CN, J app = 7.6), 2.64 (d, 1 H, H-CH=oxirane,
J app = 4.4), 2.72 (d, 1 H, F-C H =oxirane, J app = 4.3). *H NMR (200 MHz,
C 6 D6; L it.89): 0.84 (s, 3 H, C ff3-), 1.20-1.64 (m, 4 H, C # 2 CH 2 CN), 2.07 (d,
H, H-CH=oxirane, J app = 4.7), 2.13 (d,
1
1
H, H-CH=oxirane, J app = 4.7). 13C
NMR: 12.4 (C2), 20.1 (CH3), 31.6 (C3), 52.8 (CH 2 =oxirane), 54.8 (C=oxirane),
119.0 (CN). IR: 3043 (oxirane), 2250 (O N ).
(/?S)-5-(Ar,A ? -D im e th y la m in o )-4 -h y d ro x y -4 -m e th y lp e n ta n e iiitrile
(10). A solution of 11 (52 mmol, 5.8 g) in MeOH (14 mL) was added dropwise
to a solution of HNM e 2 (74 mmol, 3.34 g) in MeOH (20 mL). The m ixture
was stirre d for 1.5 h a t rt. C oncentration of the reaction m ixture gave a light
yellow oil. V acuum distillation afforded 7.30 g (90%) of a colorless oil (bp
103-104 °C/17 Torr).
!H NMR (100 MHz): 1.13 (s, 3 H, C # 3 -C), 1.60-1.90 (m, 2 H, C # 2 CH 2 CN),
2.31 (s,
2
H, CJT2 -N), 2.38 (s,
6
H, (CH 3 )2 N-), 2.40-2.60 (m,
2
H, CH 2 CN). 13C
NMR: 11.5 (C2), 24.8 (CH 3 -C), 35.6 (C3), 48.3 (CH 3 -N), 68.4 (C5), 70.5 (C4),
120.5 (CN).
IR: 3483 (OH), 2248 (C=N), 1044 (N-C).
MS m /e (relative
70
intensity): 156 (5.2), 141 (29.2), 102 (100), 87 (16.7), 59 54.2), 58 (27.6). Anal.
Calcd for C 8 H 1 6 N 2 0 : C, 61.49; H, 10.34; N, 17.93. Found: C, 61.27; H, 10.49;
N, 17.61.
(/?<Sl)-3-C yano-l-((dim ethylainino)niethyl]-l-m ethylpropyl
3,5-
dinitrobenzoate (18). A solution of 3,5-dinitrobenzoyl chloride (23 mmol,
5.41 g) in CH 2 CI2 (25 mL) was slowly added to a stirred m ixture of 10 (21
mmol, 3.28 g,) and E t3N (5 mmol, 0.50 g) in CH 2 CI2 (30 mL) a t 0 °C (icew ater bath).
A fter th e addition was complete, the reaction m ixture was
stirred for an additional 2 h a t 0 °C. The pH was adjusted to 12 w ith 5%
NaOH. The phases were sep arated and the aqueous solution w as extracted
w ith CH 2 CI2 (2 x 25 mL). The combined CH 2 CI2 solutions were w ashed w ith
H 20
(2
x 20 mL), dried, an d concentrated to give 8.05 g (100%) of a brown oil.
!H NMR (100 MHz): 1.69 (s, 3 H, CH 3 -C), 2.35 (s,
(m, 4 H, CH2CH2CN), 2.79 (d,
1
6
H, (CH 3 )2 N), 2.48-2.60
H, H-CH-N, J app = 7.2), 2.89 (d,
1
H, H-CU-
N, J app = 7.3), 9.11 (d, 2 H, o-H of benzoyl group, J app = 0.9), 9.22 (t, 1 H, p-
H of benzoyl group, J app = 1.0).
IR: 3104 (aromatic), 2249 (C=N), 1728
(C=0), 1087 (C-N).
R esolu tion o f CR)-18 and (S)-18.
Racemic m ixture of 18 was
resolved by chiral column HPLC (CHIRALCEL OD column, 25 cm x 2 cm, J.
T. B aker Inc., hexane:EtO H = 6:1, flow rate = 7 mL/min, injection size = 70 |i
g, retention time: 93 m in, 105 min), giving Cff)-18 and (SO-18.
configurations were not determ ined.
IR and *H NMR spectra are th e sam e as 18.
Absolute
71
A m in o ly sis o f 18.
HNM e 2 (45 mmol, 2.03 g) was bubbled into a
solution of 18 (21 mmol, 7.35 g) in MeOH (60 mL). The reaction m ixture was
stirred a t r t for 0.5 h an d th en concentrated. The residue was tak en up in
H 2 O (30 mL) an d filtered to remove th e lig h t yellow solid iV,iV-dimethyl 3,5dinitrobenzoate amide. The aqueous solution was extracted w ith CH 2 CI2 (3 x
20 mL). The ex tract was dried and concentrated to give a yellow oil. Vacuum
distillation afforded 2.49 g (76%) of 10 as a colorless oil.
(f2 )-3 -(l-M e th y lo x ira n y l)-2 -p ro p e n e n itrile ((U)-20). A solution of
oxalyl chloride (0.18 mol, 22.86 g) in CH 2 CI2 (180 mL) was placed in a 1000
mL 3-neck round-bottom flask equipped w ith a therm om eter, a C aS 0
4
tube,
-60
an d
a
dropping
(acetonitrile/acetone/C 0
2
funnel
and
cooled
to
-50
to
drying
°C
). A solution of DMSO (0.39 mol, 30.42 g) in CH 2 CI2
(60 mL) w as added and stirre d for 5 m in, followed by th e addition of (R)-5
(0.124 mol, 11.25 g) in CH 2 CI2 (75 mL).
S tirrin g was continued for an
additional 20 min. EtgN (0.75 mol, 76 g) was added and th e reaction m ixture
was stirred for 5 m in and th en allowed to w arm to rt. H 2 O (250 mL) was
added and th e aqueous lay er was extracted w ith CH 2 CI2 (4 x 70 mL). The
organic layers were combined, w ashed w ith satd brine (100 mL), 10% HC1 (50
mL), H 2 O (50 mL), 5% N a 2 CC> 3 (50 mL), and H 2 O (50 mL), and dried, which
was assum ed to be a solution of (S)-( l-m ethyloxiranyl)form aldehyde ((S)- 19)
an d CH 2 C12.
A solution of (E t 0 )2 P( 0 )CH 2 CN (0.135 mol, 24.03 g) in MeOH (50 mL)
was added u n d er N 2 to a stirred solution of C ^ O L i (0.135 mol) in MeOH
72
(prepared by adding 54 mL of 2.5 M solution of BuLi in hexanes into MeOH
(50 mL) a t 0 °C) m aintained a t 0 °C. Then th e solution of (S)-19 (above) was
added to th e m ixture a t 0 °C.
The reaction m ixture was stirred a t rt
overnight an d th en quenched w ith H 2 O (50 mL).
The organic phase was
separated an d w ashed w ith 10% HC1 (50 mL) and H 2 O (50 mL), dried and
concentrated.
The resu ltin g oil was purified by colum n chrom atography
(Alumina, 80-200 m esh, hexanerether = 1 : 1 0 ) to give 5.77 g (43% from CR)-5)
of a colorless oil.
!H NMR (100 MHz): 1.50 (s, 3 H, CH s-), 2.76 (d, 1 H, CH=oxirane, J app =
5.3), 2.92 (d, 1 H, CH=oxirane, J app = 5.4), 5.59 (d, 1 H, C-CH=, J app = 16.3),
6.60 (d,
1
H, =CH-CN, J app = 16.4).
IR: 3060 (olefinic C-H stretch), 2226
(CH=CH-C=N), 1634 (C=C stretch), 908 (olefinic C-H bend).
(J?) - 5 - (N, N - D im ethylam ino) - 4 - hyd roxy - 4 - m eth yl - 2 p en ten en itrile (CR)-21). A solution of CR)-20 (52 mmol, 5.67 g) in MeOH
(14 mL) w as added dropwise to a solution of HNM e 2 (74 mmol, 3.34 g) in
MeOH (20 mL). The reaction m ixture was stirred for 1.5 h. C oncentration of
th e reaction m ixture gave a light yellow oil. V acuum distillation afforded
7.10 g (89%) of a colorless oil.
!H NMR (100 MHz): 1.22 (s, 3 H, C tf 3 -C), 2.30 (s,
6
H, (CH 3 )2 N-), 2.45 (s, 2
H, -CH 2-), 5.78 (d, 1 H, C-CH=, J app = 16.0), 6.74 (d, 1 H , =CH-CN, J app =
15.8). IR: 3464 (OH), 3060 (olefinic C-H stretch), 2225 (CH=CH-C=N), 1632
(C=C stretch), 1042 (N-C).
73
Com pound (R)-10 (from CR)-21). A solution of 0R)-21 (5 mmol, 0.77
g) in absolute EtO H (20 mL) containing 10% P d on charcoal (0.3 g) was
hydrogenated a t 25 psi (pressure reaction ap p aratu s) and r t for 24 h. The
reaction m ixture w as filtered and concentrated affording a colorless oil,
which w as purified by column chrom atography (SiC>2 , 70-230 mesh,
m ethanol) (0.68 g, 87%).
IR and *H NMR spectra are th e sam e as compound 10.
(SM l-M ethyloxiranyl)m ethyl 4-n itrobenzen esu lfonate (0S)-22).
A solution of 4-nitrobenzenesulfonyl chloride (19.4 mmol, 4.3 g) in CH 2 CI2
(10 mL) w as slowly added to a stirred m ixture of (R )-2 -methylglycidol (15.3
mmol, 1.39 g, 97% purity) and E t 3 N (23.7 mmol, 2.37 g) in CH 2 CI2 (10 mL) a t
0 °C (ice-water bath). The reaction m ixture was stirred for additional 3 h a t
0 °C and th en stored in freezer for 10-12 h. The reaction m ixture was diluted
w ith cold CH 2 CI2 (10 mL) an d filtered w ith suction to remove precipitated
am m onium salt which th en w as w ashed w ith cold CH 2 CI2 (10 mL).
The
m other liquor was w ashed w ith cold 5% HC1 (10 mL), satd NaHCC > 3 (10 mL),
cold H 2 O ( 2 x 8 mL), and dried. C oncentration of th e solution gave 4.06 g of a
light yellow solid. R ecrystallization from EtO H gave 3.26 g (78 %) of (<S)-22.
1H NMR (100 MHz): 1.37 (s, 3 H, CH r ), 2.67 (d, 1 H, H-CH=oxirane, J app =
4.7), 2.72 (d, 1 H, H-CH=oxirane, J app = 4.7), 4.01 (d, 1 H, # -C H O S 0 2 Ar,
Japp = 10.9), 4.27 (d,
1
H, H -C H O S 0 2 Ar, J app = 10.9), 8.09-8.47 (m, 4 H, Bz).
CR)-2-Hydroxy-3-iodo-2-methylpropyl
4-n itrobenzen esu lfonate
((J?)-23). Compound (S)-22 (0.18 mmol, 50 mg) was added to a solution of
74
N al (0.3 mmol, 45 mg) in acetic acid (0.10 mL) and H 2 O (0.01 mL) a t 5 °C.
A fter
1
h, th e m ixture was poured into H 20 (3 mL). The aqueous solution
was extracted w ith E t20 ( 3 x 2 mL).
The combined E t20 extracts were
w ashed w ith satd NaHCC > 3 u n til n eu tral (a trace of NaHSC > 3 removed free
I2), th en w ith H 20 (3 mL), and dried. C oncentration of the solution gave a
lig h t yellow
solid.
C rystals
for X-ray
analysis
were
obtained
by
recrystallization from MeOH.
*H NMR (100 Hz): 1.41 (s, 3 H, CH s-), 3.31 (s, 2 H, ICH2-), 4.11 (s, 2 H, CH2OS), 8.10-8.49 (m, 4 H, Bz).
(2R, 6R; 2R, 6S; 2S, 6S; 2S, 6R) - 2 - (2 - C yanoethyl) - 6 -
(m ethoxyearbonyl)m ethyl-2,4,4-trim ethylm orpholinium brom ide (7).
To a stirred solution of m ethyl-4-brom o-2-butenoate (14 mmol, 2.51 g) in
CH 3 N 0 2 (30 mL) w as added 10 (14 mmol, 2.18 g) dissolved in CH 3 N 0
2
(20
mL). The solution was stirred a t 50-60 °C for 3 h. A fter cooling to rt, the
reaction m ixture w as concentrated and the residue was w ashed w ith E t 2 0 .
A fter drying u n d er vacuum , it was dissolved in MeOH (130 mL) and refluxed
for 18 h. C oncentration of th e reaction m ixture gave a brow n oil, which was
w ashed w ith eth er and dried u n d er vacuum affording 4.70 g (100%) of a
yellow oil, which was used for next reaction w ithout fu rth e r purification.
M ethyl (2S, 6R; 2R, 6 S ) - 2 - [6 - (2 - cyanoeth yl) - 4, 6 - dim ethyl
m orpholinyl] a cetate ((2S, 6R;2R, 6 S )-8) and m eth yl {2R, 6R;2S, 6’S)-2-[6(2-cyanoethyl)-4,6-dim ethylm orpholinyl]acetate
((2R, 6R; 2S, 6S)-8).
Crude 7 (14 mmol, 4.70 g) w as dissolved in A^A/'-dimethylethanolamine (20
75
mL) a t 50 °C u n d er N 2 . A fter 15 min, thiophenol (42 mmol, 4.6 g) was added
in one portion, an d th e tem p eratu re was raised to 70 °C for 24 h. The
reaction m ixture was th en cooled to rt. The pH was adjusted to 1-2 w ith 10%
HC1. The m ixture w as extracted w ith hexanes (3 x 20 mL). To th e aqueous
layer, 10% NaO H was added u n til pH~12. The aqueous layer was extracted
w ith CH 2 Cl 2 (4 x 20 mL). The combined CH 2 CI2 extracts were w ashed w ith
H 20 (2 x 20 mL) an d dried. C oncentration of the solution gave crude product
8
as a m ixture of four stereoisom ers. Separation of this m ixture by column
chrom atography (SiC>2 , 230-400 m esh, EtOAcrMeOH = 100:3) gave a solid
{(2R,6R;2S,6S)-8 , 0.36 g, m p 48.8-49.8 °C) and an oil i(2S,6R; 2R,6S)- 8 , 0.48
g) (total yield: 25% from 10). C rystals of (2R,6R;2S,6S)-8 for X-ray analysis
were obtained by crystallization from hexanes.
(2R,6R;2S,6S)- 8 :
!H NMR (400 MHz): 1.10 (s, 3 H, CH 3 -C 6 ), 1.50-1.58 (m, 1 H, H-CHCH 2 CN),
1.67 (t,
1
H, H(ax)-C3, J app = 10.8), 1.82 (d,
1
H, H(ax)-C5, J app = 11.5), 2.21
(s, 3 H, CH 3 -N), 2.32-2.52 (m, 4 H, CH 2 C 0 2 CH3, CH 2 CN), 2.47 (d, 1 H,
H(e q)-C5, J app = 11.5), 2.68 (d, 1 H, H(eq)-C3, J app = 10.9), 2.83 (m,
CHCH 2 CN), 3.70 (s, 3 H, CH 3 -O), 4.03 (m, 1 H, H- C2).
1
H, H-
^ C NMR: 10.6
(CH 2 CN), 24.6 (CH 3 -C 6 ), 29.6 (CH 2 CH 2 CN), 38.5 (CH 2 COO), 46.0 (CH 3 -N),
51.7 (CH 3 -O), 59.1 (C3), 63.9 (C5), 66.3 (C 2 ), 72.4 (C 6 ), 120.6 (CN), 171.2
(C=O). IR: 2246 (C=N), 1739 (C=0). MS m /e (relative intensity): 240 (11.6),
225 (0.5), 209 (24.0), 200 (1.1), 186 (4.9), 181 (1.1), 167 (9.1), 143 (42.7), 128
(20.8), 115 (7.0), 98 (24.1), 84 (98.2), 82 (9.4), 70 (46.7), 59 (19.9), 57 (21.1), 43
76
(100), 42 (68.2), 41 (29.2), 29 (6.5). Anal. Calcd for C 1 2 H 2 oN 2 0
3
: C, 59.98; H,
8.39; N, 11.66. Found: C, 59.79; H, 8.26; N, 11.44.
(2S,6R;2R,6S)-S:
!H NMR (400 MHz): 1.34 (s, 3 H, 6 -C # 3), 1.64 (t, 1 H, H( ax)-C3, J app = 1 0 .8 ),
1.78 (m, 3 H, H(ax)-C5, C if 2 CH 2 CN), 2.22 (s, 3 H, CH 3 -N), 2.32-2.48 (m, 5 H,
H(eq)-C5, CH 2 C 0 2 CH3, CH 2 CN), 2.75 (d, 1 H, H(eq)-C3, J app = 10.9), 3.69 (s,
3 H, C # 3 -0), 4.18 (m, 1 H, H- C2).
NMR: 11.0 (CH 2 CN), 20.3 (CH 3 -C 6 ),
36.7 (CH 2 CH 2 CN), 38.9 (CH 2 COO), 46.3 (CH 3 -N), 51.7 (CH 3 -0 ), 59.9 (C3),
62.9 (C5),
6 6 .6
(C2), 72.2 (C 6 ), 120.4 (CN), 171.2 (C=0).
IR: 2248 (C=N),
1739 (C=0). MS m /e (relative intensity): 240 (10.4), 225 (0.7), 209 (23.1),
200 (1.1), 186 (3.6), 181 (1.1), 167 (5.5), 143 (36.9), 128 (17.9), 115 (6.0), 98
(25.0), 84 (97.5), 82 (8 .6 ), 70 (37.5), 59 (17.9), 57 (17.6), 43 (100), 42 (59.2), 41
(26.6), 29 (5.6). Anal. Calcd for C 1 2 H 2 0 N 2 O3: C, 59.98; H, 8.39; N,
1 1
.6 6 .
Found: C, 59.17; H, 8.73; N, 11.08.
D e te r m in a tio n o f o p tic a l p u r it y o f 2 -m e th y lg ly c id o l b y
NM R. CR)-(+)-MTPA (2.05 g), S0C1 2 (4 mL) and NaCl (0.03 g) were refluxed
for 50 h. The excess S0C1 2 was removed by vacuum evaporation and the
residue w as distilled to give CR)-(+)-MTPA-Cl. 8 2
To a solution of 2-methylglycidol (0.15 mmol, 13.2 mg) an d E t3N (0.06
mL) in CH 2 C12 (1 mL) was added (i?)-(+)-MTPA-Cl (0.10 mL). After being
allowed to stan d in refrigerator (0 °C) for 10 h, the reaction m ixture was
w ashed w ith 5% H 2 SC> 4 (2 mL), filtered, and concentrated affording the
M osher's ester.
77
1H NMR (200 MHz):
(a) M osher's ester of (R)-(+)-2-methylglycidol: 1.36 (s, 3 H, C-CH 3 ),
2.64 (d, 1 H, H -CH =oxirane, J app = 4.6), 2.77 (d,
1
H, H- CH=oxirane, J app =
4.6), 3.56 (s, 3 H, OCH3), 4.23 (d, 1 H, ff-CHO-MTPA, J app = 11.9), 4.47 (d,
1
H, H-CHO-MTPA, J app = 11.8), 7.35-7.68 (m, 5 H, arom atic).
(b) M osher's ester of (<S)-(-)-2-methylglycidol: 1.33 (s, 3 H, C-CH 3 ), 2.64
(d, 1 H, H-CH =oxirane, J app = 4.7), 2.74 (d, 1 H, H-CH=oxirane, J app = 4.7),
3.56 (s, 3 H, OCH 3 ), 4.16 (d, 1 H, H-CHO-MTPA, J app = 11.8), 4.50 (d, 1 H,
H-CHO-MTPA, J app = 11.9), 7.35-7.68 (m, 5 H, aromatic).
The optical purities of (R )- and (S)-2-methylglycidols were determ ined
by in teg ratio n of th e signals (dd) of -i^C O -W -M T PA .
The ee values are
88.4% for (R )-2-methylglycidol an d 91.8% for (S)-2-methylglycidol.
D e te r m in a tio n o f o p tic a l p u r it y o f c o m p o u n d 13 b y *H N M R. A
solution of 13 (3 mmol, 0.711 g) in MeOH (9 mL) was added to a solution of
HNM e 2
(1 2
mmol, 0.54 g) in MeOH (3 mL). The reaction m ixture was stirred
for 2 h an d th en concentrated. MeOH (3 mL) w as added, an d the precipitate
w as rem oved by filtration. T he solution w as concentrated. Again, MeOH (2
mL) w as added, and th e precipitate was rem oved by filtration. The solution
w as placed in freezer for 0.5 h. The liquid was pipetted out and concentrated
by ro ta ry evaporation and th e n placed u n d er vacuum . V acuum distillation of
th e residue gave 0.30 g (75%) of 3-dim ethylam ino-2-m ethylpropane-l,2-diol,
6
, as a colorless oil (bp: 150 °C/17 Torr). ^H NMR (200 MHz): 1.07 (s, 3 H,
CH 3 -C), 2.37 (s,
6
H, CH 3 -N), 2.43 (dd, 1 H, H-CHN, J app = 13.7, 1.8), 2.57
78
(d, 1 H, H-CHN, J app = 13.6), 3.46 (dd, 1 H, H-CHOH, J app = 11.0, 1.7), 3.67
(d, 1 H, H-CHOH, J app = 1 1 . 1 ). 13C NMR: 23.7 (CH 3 -C), 47.9 (CH 3 -N), 67.9
(-CH 2 -N), 70.7 (-CH 2 -OH), 71.2 (C-OH).
IR: 3405 (OH), 1044 (C-N). MS,
FAB, m/e: 134 (M+ + 1). Anal. Calcd for C 6 H 1 5 N 0 2: C, 54.14; H, 11.28; N,
10.53. Found: C, 53.82; H, 11.10; N, 10.41.
To a solution of 6 (0.18 mmol, 24 mg) and E t3N (0.05 mL) in CH 2 C12 (1
mL) was added CR)-(+)-MTPA-Cl (0.10 mL) a t 0 °C. The reaction m ixture was
placed in a freezer for 10 h. N aO H solution (5%) was added u n til pH ~ 1 2 .
The phases were separated an d th e aqueous solution was extracted w ith
CH 2 C12 ( 3 x 2 mL). The combined CH 2 C12 extracts were w ashed w ith H 20 (2
x 5 mL), dried, an d concentrated to give M osher’s ester, 15, as a n oil.
The optical p u rities of (R )- and (SO-13 were determ ined by integration
of th e *H NMR signals of CH 3-C of M osher's ester, 15. For optical enriched
13, th e m inor isom er was undetectable.
!H NMR (400 MHz): 1.086 (s, CH 3 -C) for M osher’s ester of (S)-6 , 1.069 (s,
CH 3 -C) for M osher's ester of (R)-6 .
C h a p te r
III
INHIBITION OF CAT BY FOUR STEREOISOMERS
OF 6-CARBOXYLATOMETHYL-2HYDROXYMETHYL-2,4,4-TRIMETHYL
MORPHOLINIUM
The four stereoisom ers of 6-carboxylatomethyl-2-hydroxym ethyl-2,4,4trim ethylm orpholinium , II, w ere tested as specific inhibitors of CAT by Drs.
Noirfn Nic a' B haird an d Rona R. Ram say a t UCSF.
The resu lts are
presented an d discussed in th is chapter.
III.l. PR EVIO US WORK
Pigeon b re a st CAT indiscrim inately binds both enantiom ers of
carnitine an d acetylcarnitine , 1 0 3 >1 0 4 which indicates a two-point recognition
of carnitine, involving carboxylate and trim ethylam m onium groups.
Cff)-
C arnitine an d (R )-acetylcarnitine are su b strates in the forw ard an d reverse
reactions
respectively,
w hile
th e
S
enantiom ers
are
competitive
inhibitors. 103,104 This stereospecificity indicates th a t th e location of th e CoA
binding site m u st be closer to th e hydroxyl on (R)-carnitine th a n th e one on
(S)-carnitine. (Figure I I I .l ) . 7 2
79
80
C oA
CoA
HS
OH
CH
Me'
•N +
II
Me
Me
Me
(/?)-carnitine + acetyl-C oA
(/?)-acetylcarnitine + C oA
CoA
CoA
HS
HO '
Me'
Me
Me
(5')-carnitine + acetyl-C oA
(,S')-acetylcarnitine + CoA
F igure III.1 . Mode of su b strate recognition in CAT
CoAS
CoA
.()—
Me
Me
ON—Me
/ \
Me
Me
Me
tetrahedral intermediate
r ^OH
N -M
S c h e m e I I I .l
Me
analogue inhibitor
81
Based on the hypothesis for the mode of su b strate recognition in CAT,
a m echanism for th e reversible acetyl tra n sfer betw een carnitine and CoA
h as been proposed (Scheme I I I .I ) . 7 2 In th is m echanism , the thiolate attacks
on th e Re face of th e este r forming R configuration on th e tetrah ed ral
in term ed iate carbon.
K{ (|HM)
24. R = CH2C 0 2H
530
25. R = CH 3 (racemic)
1080
26. R = H (racemic)
1000
F ig u r e III.2. T e trah ed ral interm ediate analogues and
th e ir A- values w ith respect to CR)-carnitine
Three tetra h ed ral-in term ed iate analogues , 1 0 5 synthesized and assayed
w ith CAT in 1987, (Figure III.2) m u st have th e sam e configuration a t C 6 as
(-R)-carnitine does a t C3 to effectively in h ib it CAT. O f these analogues, 24
binds m ost strongly, w ith a K\ h a lf th a t of the racem ic compounds 25 and 26.
Because every molecule of 24 has one side th a t correctly m atches the
configuration of (jR)-carnitine an d only h a lf of th e molecules of racem ic 25
an d 26 have th e
correct configuration
of (i?)-carnitine, th e
twofold
im provem ent in binding for 24 suggests th a t CAT is selectively binding one
configuration of these inhibitors more tightly th a n the others (chiral
recognition of C 6 ).
To confirm th is speculation, more direct and potent
82
evidence is needed. In addition, those analogues do not te s t th e configuration
of th e te tra h ed ral in term ediate carbon (C2 in the analogues) because 24 is a
meso (R S ), 25 is a (R S \S R ) racem ate, and 26 is a (R;S) racem ate. Inhibition
studies w ith th e four stereoisom ers of I I provide a te s t of th e configuration
stereocenters.
III.2. R E S U L T S AND D IS C U S S IO N
The concentrations of th e inhibitors needed for 50% inhibition (IC 5 0 ) of
commercial pigeon b reast CAT have been determ ined a t 250 p.M of (R )acetylcarnitine (Km = 350 |iM). The d ata are shown in Table III.l. All the
four stereoisom ers of I I in h ib it CAT to varying degrees. Compound (2S,6R)II is th e m ost potent inhibitor of CAT am ong these four stereoisom ers. The
value of K\ for th is stereoisom er shows th a t it binds 2-fold b etter th a n (R)acetylcarnitine to CAT.
The d a ta in Table III.l also indicate th a t CAT
recognizes both th e configurations of C 6
and C2 in the tetrah ed ral-
in term ed iate analogues.
Compound (2S,6R )-II binds 10-fold b etter th a n (2S,6S )-II to CAT.
Both of them have th e sam e configuration of C2.
binds 2.5-fold b ette r th a n C2f?,6S)-II to CAT.
Compounds (2R,6R )-II
Both of them also have the
sam e configuration of C2. The difference in both comparisons of inhibition of
CAT is th e difference of configurations of carnitine fragm ents (C 6 ). These
com parisons confirm th e previous conclusion th a t CAT recognizes the
configuration of C 6 and strongly prefers R configuration.
83
T a b le I I I .l. Inhibition of 6 -carboxylatom ethyl- 2 -hydroxymethyl2,4,4-trim ethylm orpholinium on CAT
Inh ib itor
S tructure
I C 50 ( m M )
A'j ( g M )
OH
,Me
0.42
(2S, 6R)-Yl
Me
187
Me
OH
.Me
1.36
( 2R, 6R)-n
Me
Me
'OoC
,O H
H
Me
(.2R, 6S)-1l
3.41
Me
Me
^,OH
' 0 0C
H
(2S,
/
6S)-II
0 ^ 1 Ale
4.04
I
; Nv
Me
Pigeon
b re ast
CAT
binds
acetylcarnitine equally w ell . 1 0 3 ’ 1 0 4
Me
enantiom ers
of both
carnitine
and
B ut only (R )-enantiom ers can undergo
acetyl tran sfer, which m eans th a t only CR)-enantiomers can form a
tetrah ed ral-in term ed iate. W hen the tetrah ed ral-in term ed iate forms (acetyl-
84
CoA is attach ed to carnitine), the enzyme is probably in a different
conformation th a n w hen it binds carnitine or acetylcarnitine. This enzymic
conform ation com plem ents only the CR)-carnitine tetrahedral-interm ediate.
Therefore, th e enzym e in th is conformation should be inhibited by the
reaction-interm ediate analogues th a t have the sam e configuration as (R)carnitine (C 6 ).
This speculation agrees w ith our results, confirming the
hypothesis th a t th e enzymic conformation for binding these analogues is
different th a n th e enzymic conform ation th a t binds th e substrates.
Com pounds (2S,6R )-II an d (2R,6R )-II have the sam e configuration on
C 6 b u t different configurations on C 2 . Compound (2S,6R )-II binds 3-fold
b etter th a n (2R,6R )-II to CAT, suggesting th a t S configuration of C2 is
required for enzyme recognition.
This resu lt strongly supports the
m echanism for acetyl tra n sfe r betw een carnitine an d CoA proposed by
G andour e t al ? 2 (Scheme III.l). The idea is th a t th e thiolate approaches the
acyloxy from th e less-congested side and attacks on th e Re face of the ester to
form
R
configuration
configuration
in
th e
on
th e
analogue
S
tetra h ed ral-in term ed iate
(C5).
The
inhibitor
sam e
relative
(C2) is
th e
configuration as th e R configuration for th e tetra h ed ral-in term ed iate (C5).
This idea agrees w ith th e observation th a t C2»S,6R)-II is the best inhibitor
am ong four stereoisom ers.
Com pared w ith th e previously synthesized analogue inhibitors 24, 25,
and 26, which are meso an d racemic compounds and do not te st the
configuration of te tra h e d ra l in term ediate carbon (C2 in the analogues), our
analogues are chiral compounds so th a t they can te st not only the
configuration of carnitine fragm ent b u t also the configuration of tetra h ed ral
in term ediate carbon.
Compounds C2£,6i?)-II, 24, an d th e more active
enantiom ers of 25 an d 26 have the same configuration on C 6 . Racemic 25
and 26 have K\ values of 1080 and 1000 respectively. We assum e th a t the
more active enantiom ers of 25 and 26 have K j values of 540 and 500
respectively.
Compound (2S,6R )-II binds nearly 3-fold b etter th a n 24 and
th e active enantiom ers of 25 and 26 to CAT. The possible reasons for these
resu lts are th a t (1) th e configuration of C2 in (2S,6R)-Il fits the enzyme
m uch b e tter th a n th e configurations of C2 in 24, 25, and 26 do, and (2)
hydrogen bonding is im p o rtan t for (2,S,6R)-II.
By com paring th e IC 5 0 values of (2S,,6i?)-II w ith (2R,6R )-II and
(2S,6R)-11 w ith (2S,6S)- II, we can see th a t the configuration of C 6 is more
crucial th a n th a t of C 2 . We speculate th a t C 6 in th e analogue mimics C3 in
the tetrah ed ral-in term ed iate b etter th a n C 2 in th e analogue mimics C5 in
th e in term ed iate (Scheme III.l).
Carbon
6
in the analogue and C3 in the
interm ediate have alm ost th e sam e environm ent, while in the interm ediate
C5 is connected to CoA and a negatively charged oxygen and in th e analogue
C2 is in a six-m em bered rin g and connected to a hydroxym ethyl group. This
difference m ight affect enzyme recognition. In addition, th e binding of CoA
to CAT is different from the interaction of hydroxym ethyl w ith CAT.
Therefore, th e im portance of th e configuration of C5 in the tetrah ed ralinterm ediate m ight be underestim ated by th is inhibitory study.
III-3. C O N C L U SIO N
In conclusion, th e re su lts of the study verify firm ly th a t the enzyme
recognizes both configurations of C2 an d C 6 in the tetrahedral-interm ediate
analogues.
If th e enzyme hinds these analogue inhibitors as it binds the
tetrah ed ral-in term ed iate, we can conclude th a t th e enzymic conformation for
binding th e su b strates is different from th e enzymic conform ation th a t binds
the tetrah ed ral-in term ed iate.
CAT binds both R and S enantiom ers of
carnitine or acetylcarnitine b u t the chiral center of carnitine is not
recognized. The chiral recognition of carnitine is caused by the binding of
CoA. The thiolate of CoA attack s on th e Re face of th e ester of carnitine to
form R configuration on th e te tra h ed ral interm ediate carbon. Only the
tetrah ed ral-in term ed iate w ith R configurations on C3 and C5 forms in the
acetyl tra n sfe r reaction betw een carnitine and CoA.
enantiom ers can undergo acetyl transfer.
Therefore, only (R)-
C h a p te r
IV
CONCLUSION
IV. 1. R E S U L T S
We have developed new synthetic m ethods and added new m em bers to
th e fam ily of tetrah ed ral-reaction-interm ediate analogue inhibitors.
The
inhibitory effects of th ese new analogues on CAT have been studied.
1.
We have p rep ared four stereoisom ers of
6
-carboxylatom ethyl- 2 -
hydroxym ethyl-2, 4, 4-trim ethyl m orpholinium , II, which are reactionin term ed iate analogue inhibitors and will serve as precursors to four
stereoisom ers
of
2-(3-aminopropyl)-6-carboxylatomethyl-2,4,4-trim ethyl
m orpholinium , III, an d th e ta rg e t molecules of the project. This is th e first
tim e th a t four stereoisom ers of th is type of acyltransferase inhibitors are
m ade.
2
. We have prepared m ethyl (2S,6S;2R,6R )- and (2S,6R',2R,6S)-2-[6-
(2-cyanoethyl)-4,6-dim ethylm orpholinyl]acetate,
8
, which are precursors to
molecules III.
3.
We have resolved racem ic m ixture of 5-(N,N-dimethylamino)-4-
hydroxy-4-m ethylpentanenitrile, 10, which m akes it possible th a t four
87
88
stereoisom ers
of
2-(3-aminopropyl)-6-carboxylatomethyl-2,4,4-trim ethyl
m orpholinium , III, be m ade sta rtin g w ith nonchiral compound, 3-methyl-3buten-l-ol.
4.
of CAT.
The four stereoisom ers of I I have been tested as specific inhibitors
The resu lts confirm the hypothesis for th e mode of substrate
recognition in CAT and th e m echanism for acetyl tra n sfer betw een carnitine
and CoA.
A lthough I did not get 2-(3-aminopropyl)-6-carboxylatomethyl-2,4,4trim ethylm orpholinium , III, due to various reasons, I came relatively close
by preparing four stereoisom ers of 6-carboxylatom ethyl-2-hydroym ethyl-2, 4,
4-trim ethyl m orpholinium , II, which are precursors to III, and resolving
racemic
m ixture
m ethylpentanenitrile,
1 0
of
5-(Ar,A’- dimethylamino)-4-hydroxy-4-
, w hich m akes it possible th a t four stereoisom ers of
I II be m ade sta rtin g w ith nonchiral compound, 3-m ethyl-3-buten-l-ol.
Furtherm ore, compound II can lead to other compounds th a t m ight be potent
inhibitors, (see below)
IV.2. F U T U R E D IR E C T IO N
The fu tu re work will be directed tow ard th e synthesis of four
stereoisom ers
of
2-(3-aminopropyl)-6-carboxylatomethyl-2,4,4-trim ethyl
m orpholinium , III, which are precursors to th e ta rg e t molecules of the
project.
89
According to th e resu lts of our work (section IV. 1), th ere are a num ber
of possible synthetic routes for III, w hich are shown in Scheme IV. 1 and
Scheme IV. 2.
Scheme IV. 1 is a continuation of Scheme 11.15.
eyanoethyl)-4,6-dim ethylm orpholinyl]acetate,
8
, can
be
M ethyl 2-[6-(2converted
into
compound III by m ethylation of te rtia ry am ine, hydrolysis of m ethyl ester,
and reduction of cyano group. The sequence of those th re e reactions m ight be
crucial to th is conversion. It is not difficult to find out th e correct sequence.
Four stereoisom ers of
8
can be m ade from 3-m ethyl-3-buten-l-ol and optical
resolution of (R S )-5-(A^,A’-dim ethylam ino)-4-hydroxy-4-m ethylpentanenitrile,
10, is needed.
COOMe
COOMe
COO
S c h e m e IV. 1
In Scheme IV.2, compound I I I can be m ade in a num ber of ways. In
these synthetic routes, th ree more analogue inhibitors, 27, 30, and 33, can be
m ade in addition to III. In synthetic route of Scheme IV. 1, only one more
analogue inhibitor, 27, can be m ade in addition to III.
Furtherm ore,
compounds 1, 2, an d II in Scheme IV.2 are m ade startin g w ith chiral
compound (R )- or (S)-2-methylglycidol (Scheme II. 6 ), no optical resolution is
needed. Therefore, Scheme IV.2 will be b e tte r th a n Scheme IV. 1.
COOMe
OH
COOMe
OH
NaOH
h 2o
(2R, 6S)-2
COOMe
COOM e
(.
O
2R, 65) - !
C OOM e
.CN
C OOM e
32
COOM e
C OOM e
S c h e m e IV.2
‘
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D erivatives of Hom ochiral Glycidol: V ersatile C hiral Building Blocks
for O rganic Synthesis." J. Org. Chem. 1989, 54, 1295-1304.
92.
Johnson, C. R.; D u tra, G. A. "Reaction of Lithium Diorganocuprates(I)
w ith Tosylates. I. Synthetic Aspects." J. Am. Chem. Soc. 1973, 95,
7777-7782.
93.
Sun,
G.;
Fronczek,
F.
R.;
G andour,
R.
D.
"(RM lM ethyloxiranyl)m ethyl 4-Nitrobenzenesulfonate." Acta Crystallogr.,
Sect. C, 1992, 48, 758-760.
94.
Sun, G.; Fronczek, F. R.; G andour, R. D. "(R)-2-Hydroxy-3-Iodo-2M ethylpropyl 4-N itrobenzenesulfonate." Acta Crystallogr., Sect. C,
1993, 49, 1507-1509.
95.
Gajewski, J. J; Gilbert, K. E. PCMODEL Molecular Modeling Package,
version 4.0. 1992. S erena Software, PO Box 3076, Bloomington, IN
47402, USA.
96.
W heland, G. W. Advanced Organic Chemistry, 3rd Edt; Jo h n Wiley:
New York. 1960; p 660.
97.
Colucci, W. J. "Conformational Studies of C arnitine and
A cetylcarnitine: Design and Synthesis of Active-Site Probes of
C arn itin e A cetyltransferase." Dissertation, C hem istry D epartm ent,
Louisiana S tate U niversity, 1987.
98.
V aughan, W. R.; B aum ann, J. B. "Reaction of Alkyl Carboxylic E sters
w ith M ercaptides." J. Org. Chem. 1962, 27, 739-744.
99.
S heehan, J. C.; Daves, J r. G. D. "Facile Alkyl-Oxygen E ster Cleavage."
J. Org. Chem. 1964, 29, 2006-2008.
100. Sun, G.; Fronczek, F. R.; G andour, R. D. "Methyl (2S,6S;2R,6R)-2-[6(2-Cyanoethyl)-4,6-Dim ethylmorpholinyl]acetate." Acta Crystallogr.,
Sect. C, 1994, 50, in press.
102
101.
S erena Software. GMMX (GLOBAL-MMX, version 1.0), Steric Energy
M inimization Program. 1992.
Serena Software, PO Box 3076,
Bloomington, IN 47402, USA.
102.
P aquette, L. A.; S auer, D. R.; Cleary, D. G.; K insella, M. A.; Blackwell,
C. M.; Anderson, L. G. "Application of Palladium -catalyzed [3 + 2]
Cycloaddition Technology to the E laboration of Kem pane D iterpenes.
Stereocontrolled Synthesis of (±)-3a-Hydroxy-7P-kemp-8(9)-en-6-one
an d (±)-3P-Hydroxykemp-7(8)-en-6-one." J. Am. Chem. Soc. 1992, 114,
7375-7387.
103.
Chase, J. F. A.; Tubbs, P. K. "Some Kinetic Studies on M echanism of
Action of C arnitine A cetyltransferase." Biochem. J. 1966, 99,32-40.
104.
Tipton, K. F.; Chase, J. F. A. "The Effects of S u b strates on the Optical
Rotatory D ispersion of C arnitine Acetyltransferase." Biochem. J. 1969,
115, 517-521.
105.
Colucci, W. J.; G andour, R. D.; Fronczek, F. R.; Brady, P. S.; Brady, L.
J. "A "Siameso” Inhibitor: C hiral Recognition of a Prochiral B ilaterally
Sym m etric Molecule by C arnitine Acetyltransferase." J. Am. Chem.
Soc. 1987, 109, 7915-7916.
A p p e n d ix A
CRYSTALLOGRAPHIC DATA FOR
( 2 S , 6 R ) -2 -HYDROXYMETH YL-6(METHOXY CARBONYL)METHYL-2,4,4TRIMETHYLMORPHOLINIUM IODIDE
C 1 1 H 2 2 NO 4 I, Mr = 359.2, orthorhombic, P 2 ]2 ]2 i, a = 9.5519(6), 6 =
12.4000(6), c = 25.723(2) A, V = 3046.7(6) A3, Z = 8, Dx = 1.564 g cm’3 a t 293
K, X (Mo K a
) =
0.71073 A,
|li
=
2 0 .8
cm '1, F ( 0
0 0
) = 1440, 6251 unique data
m easured, final R = 0.027 for 5230 reflections w ith I > 3o(I).
independent form ula un its in asym m etric unit.
Table A .l. Coordinates and isotropic therm al p aram eters
Ba] = (8 tt 7 3 ) 1 1 ^ ; >ai a <a j
> 1
Atom
11
12
O lA
02A
03A
04A
N1A
CIA
C2A
y
X
0.23568(3)
0.68574(3)
0.2750(3)
0.4204(3)
0.1908(3)
-0.0145(3)
0.2419(3)
0.3112(4)
0.2556(4)
(Table con'd.)
0.21429(2)
0 .2 1 2 1 1 ( 2 )
0.2618(2)
-0.0606(2)
-0.0369(2)
0.3600(2)
0.1345(2)
0.0927(3)
0.1479(2)
103
z
0.59731(1)
0.08180(1)
0.17058(9)
0.2416(1)
0.2450(1)
0.0912(1)
0.0766(1)
0.1253(1)
0.1737(1)
B eq (A2)
3.927(5)
4.820(6)
3.03(5)
5.49(8)
5.71(8)
4.07(6)
2.73(5)
2.91(7)
2.80(6)
Two
104
Atom
C3A
C4A
C5A
C6 A
C7A
C8 A
C9A
C1 0 A
C1 1 A
01B
02B
03B
04B
N IB
C1B
C2B
C3B
C4B
C5B
C6 B
C7B
C8 B
C9B
C10B
CUB
X
0.2125(4)
0.2537(4)
y
z
0.3136(2)
0.2561(2)
0.0904(3)
0.0964(3)
0.1103(3)
-0.0029(3)
0.4251(3)
0.3232(3)
-0.1699(4)
0.2319(2)
0.5479(2)
0.1262(1)
0.0762(1)
0.0308(2)
0.0720(2)
0.2205(2)
0.2361(2)
0.1238(2)
0.1366(2)
0.2595(3)
0.33758(9)
0.2442(1)
0.5376(2)
0.0669(2)
0.3398(2)
0.3243(1)
0.4380(1)
0.4377(10
0.3857(1)
0.3446(1)
0.3836(1)
0.4305(1)
0.4750(2)
0.4601(2)
0.3507(6)
0.1047(5)
0.3949(3)
0.3378(3)
0.1651(3)
0.2234(3)
0.3914(3)
0.3520(3)
0.3948(3)
0.5001(3)
0.0704(3)
0.1247(3)
0.0913(6)
0.6495(3)
0.3191(5)
0.0934(4)
0.3391(4)
0.3057(4)
0.2765(5)
0.0553(4)
0.4029(7)
0.2585(3)
0.1613(4)
0.0742(3)
0.0916(3)
0.2739(3)
0.2860(4)
0.2037(4)
0.2533(4)
0.3165(4)
0.3756(5)
0.1320(4)
0.2186(5)
0.1427(4)
0.2922(1)
0.2906(1)
0.3724(2)
0.3906(2)
0.2366(2)
B eq (A2)
2.99(7)
2.87(6)
3.84(8)
3.54(8)
3.51(8)
3.53(8)
4.45(9)
3.49(8)
8.9(2)
3.65(5)
4.86(7)
4.03(6)
4.10(6)
2.83(6)
3.29(7)
3.13(7)
3.29(7)
3.13(7)
3.95(8)
3.56(8)
4.10(9)
3.24(8)
5.1(1)
3.72(8)
5.0(1)
105
T a b le A.2. Coordinates and isotropic therm al p aram eters
for hydrogen atom s
Atom
H 40A
H lA a
H lA b
H2A
H4Aa
H4Ab
H5Aa
H5Ab
H5Ac
H 6 Aa
H 6 Ab
H 6 Ac
H7Aa
H7Ab
H9Aa
H9Ab
H9Ac
HlOAa
HlOAa
H llA a
x
y
z
-0.083(6)
0.4090
0.2941
0.1589
0.1952
0.322(4)
0.1051
0.0174
0.1307
0.2825
0.3483
0.4141
0.2740
0.1128
0.0138
0.1164
0.087(2)
0.1229
0.1280
0.1766
0.0491
0.0688
0.0322
0.0313
-0.0003
0.1005
0.0404
0.0721
0.2489
0.3148
0.2775
0.0405
0.0541
0.0914
0.3191
0.4359
0.3741
0.2338
0.2618
0.0192
0.0400
0.4916
0.3613
0.3441
H llA b
H llA c
H 40B
0.063(3)
H IB a
0.3817
H IB b
0.2520
H2B
0.1085
H4Ba
0.2896
0.4154
H4Bb
H5Ba
0.4679
(T a b le con'd.)
0.1228
0.1223
0.0197
0.1567
0.1145
0.4191
0.4649
0.4610
0.2545
0.3729
-0.1992
-0.2119
-0.1705
0.007(2)
0.3964
0.4666
0.3367
0.1849
0.2213
0.3848
0 . 2 1 2 2
0.1173
0.0965
0.1559
0.1461
0.1641
0.2682
0.2327
0.2892
0.430(1)
0.3756
0.3887
0.3553
0.4608
0.4270
0.4617
Bjso (A2)
1 0
(2 )
3
3
3
3
3
4
4
4
4
4
4
4
4
5
5
5
4
4
1 1
1 1
1 1
1.9(6)
4
4
4
4
4
5
106
Atom
x
y
z
H5Bb
H5Bc
H 6 Ba
H 6 Bb
H 6 Bc
H 7Ba
0.3528
0.3698
0.0652
0.1282
0.4655
0.3562
0.4790
0.5077
0.4373
0.4929
0.4642
0.2658
H7Bb
H9Ba
H9Bb
H9Bc
HlOBa
HlOBb
H llB a
H llB b
H llB c
0.1113
0.1819
0.3151
0.4436
0.3478
0.3212
0.0424
0.0813
0.1191
0.1155
-0.0071
0.3201
0.3172
0.4264
0.3492
0.4076
0.0961
0 . 0 2 1 1
0.2858
0.3680
0.4006
0.0349
0.1844
0.0783
0.6797
0.6974
0.6383
0.3415
0.3910
0.3625
0.2043
0.2640
0.2365
Biso (A2)
5
5
4
4
4
5
5
6
6
6
4
4
6
6
6
A p p e n d ix
B
CRYSTALLOGRAPHIC DATA FOR
(2R,6R)-2-HYDROXYMETHYL-6(METHOXY CARBONYL)METHYL-2,4,4TRIMETHYLMORPHOLINIUM IODIDE
C 1 1 H 2 2 NO 4 I, M r = 359.2, orthorhombic, P2^2^2-\_, a = 7.9611(4), b =
13.6603(5), c = 14.0588(9) A, V = 1528.9(2) A3, Z = 4, Dx = 1.561 g c n r3 a t 291
K, X (Mo K a
) = 0.71073 A, |u. = 20.7
cm-1, 1 ^(0
0 0
) = 720, 6685 unique data
m easured, final R = 0.038 for 4863 reflections w ith I > 3a(I).
107
108
T a b le B .l. Coordinates and isotropic therm al param eters
'
Atom
I
01
02
03
04
N
Cl
C2
C3
C4
C5
C6
C7
C8
C9
CIO
C ll
X
0.72283(3)
0.3997(3)
0.7953(4)
0.5515(4)
0.3931(5)
0.1985(3)
0.3680(4)
0.4101(4)
0.2380(4)
0.1844(5)
0.1853(6)
0.0603(5)
0.5891(4)
0.6372(5)
0.1120(6)
0.2695(6)
0.8611(7)
j
y
0.19805(2)
0.3314(2)
0.5316(2)
0.5008(2)
0.1299(2)
0.3646(2)
0.4099(2)
0.4228(2)
0.2864(2)
0.2738(2)
0.3332(3)
0.4365(3)
0.4576(2)
0.4982(2)
0.3362(4)
0.1829(3)
0.5730(3)
z
0.31553(2)
0.1367(2)
0.0871(2)
0.0135(2)
0.1514(2)
0.3058(2)
0.2904(2)
0.1850(2)
0.1415(2)
0.2448(3)
0.4074(3)
0.2885(3)
0.1780(2)
0.0828(3)
0.0771(3)
0.1018(3)
0.0005(3)
B eq (A2)
4.409(4)
3.02(4)
4.66(5)
4.56(6)
5.14(7)
3.11(5)
2.87(5)
2.81(4)
3.51(6)
3.62(6)
4.36(7)
4.60(8)
3.10(5)
3.32(6)
5.7(1)
4.56(7)
5.87(9)
109
T a b le B.2. Coordinates and isotropic therm al param eters
for hydrogen atoms
Atom
X
y
z
H 40
H la
H lb
H2
H 4a
0.465(9)
0.4506
0.3699
0.3335
0.0704
0.2528
0.2715
0.1972
0.169(4)
0.3689
0.4722
0.186(5)
0.3168
0.3202
0.1573
0.2453
0.2723
0.4211
H4b
H 5a
H5b
H5c
H 6a
H6b
H6c
H 7a
H7b
H 9a
H9b
H9c
H lO a
HlOb
H lla
Hllb
Hllc
0.0788
0.0645
-0.0449
0.0734
0.6609
0.6054
0.1567
0.0109
0.0891
0.1671
0.3039
0.9784
0.4678
0.2531
0.2243
0.2872
0.3886
0.3038
0.4582
0.4061
0.4909
0.4038
0.5074
0.3405
0.2992
0.8080
0.4001
0.1473
0.1888
0.5826
0.6341
0.8390
0.5297
0.4476
0.4179
0.2243
0.3002
0.3298
0.1916
0.2242
0.0146
0.0758
0.1004
0.1047
0.0373
0.0058
-0.0112
-0.0509
Biso (A2)
11(2)
3
3
3
4
4
5
5
5
5
5
5
4
4
7
7
7
5
5
7
7
7
A p p e n d ix C
CRYSTALLOGRAPHIC DATA FOR
(2R,6S)-6-CARBOXYLATOMETHYL2-HYDROXYMETHYL-2,4,4TRIMETHYLMORPHOLINIUM
C ioH i9N 0 4.2 H 20 , Mr = 253.3, monoclinic, P2h a = 7.8558(6), b = 9.8998(7),
c = 8.6266(5) A, P = 101.314(5)°, V = 657.9(2) A3, Z = 2, Dx = 1.278 g cm '3 a t
294 K, X (Cu K a ) = 1.54184
A,
|i
= 8.45 c m '1, F(000) = 276, 2641 unique
d ata m easured, final R = 0.0369 for 2571 reflections w ith I > 3a(I).
110
Ill
T a b le C .l. Coordinates and isotropic therm al param eters
=(8jr2/ 3 ) i ; i U ija;a;alaj
'
Atom
01
02
03
04
N
Cl
C2
C3
C4
C5
C6
C7
C8
C9
CIO
01W
02W
X
0.5734(1)
0.2195(2)
0.4153(2)
0.7828(2)
0.9454(2)
0.8128(2)
0.6536(2)
0.6790(2)
0.8600(2)
1.0877(2)
1.0276(2)
0.5177(2)
0.3719(2)
0.5914(2)
0.6772(2)
0.2699(2)
0.1097(2)
J
y
0
-0.2240(2)
-0.3195(2)
0.0209(2)
-0.0086(2)
-0.0613(2)
-0.1108(2)
0.0589(2)
0.0943(2)
0.0633(2)
-0.1237(2)
-0.1685(2)
-0.2420(2)
0.1921(2)
-0.0322(2)
0.1030(2)
-0.0590(3)
z
0.7318(1)
0.5232(2)
0.7085(2)
1.1530(1)
0.7484(2)
0.6105(2)
0.6667(2)
0.8696(2)
0.8384(2)
0.6876(2)
0.8512(2)
0.5331(2)
0.5931(2)
0.8928(2)
1.0131(2)
0.0995(2)
0.2768(2)
B eq (A2)
2.99(2)
4.03(3)
4.48(3)
4.10(3)
2.86(2)
2.90(3)
2.79(3)
2.70(3)
2.98(3)
4.11(4)
3.71(3)
3.24(3)
2.92(3)
3.26(3)
3.21(3)
4.77(3)
9.42(5)
112
T a b le C.2. C oordinates and isotropic th erm al p aram eters
for hydrogen atom s
Atom
X
H 40
H la
H lb
H2
H 4a
H4b
0.713(4)
0.875(2)
0.785(2)
0.687(2)
0.838(3)
0.944(2)
H 5a
H5b
H5c
H 6a
H6b
H6c
H 7a
H7b
H 9a
H9b
H9c
H lO a
1.025(3)
1.168(3)
1.143(3)
0.932(2)
0.576(3)
0.471(3)
0.594(3)
0.471(3)
0.656(3)
0.554(2)
HlOb
H1W1
H2W1
H1W2
0.725(3)
0.373(3)
0.225(3)
0.139(5)
H2W2
-0.008(4)
1.086(3)
1.109(3)
y
0.047(5)
-0.133(2)
0.018(2)
-0.179(2)
0.170(2)
0.104(2)
0.132(3)
0.095(2)
z
1.218(4)
0.564(2)
0.535(2)
0.749(2)
0.762(3)
0.940(2)
-0.174(3)
-0.084(3)
-0.230(3)
-0.104(2)
0.259(3)
0.177(3)
0.227(3)
-0.032(2)
-0.125(2)
0.618(3)
0.777(3)
0.626(3)
0.891(2)
0.786(3)
0.933(3)
0.482(3)
0.458(2)
0.804(3)
0.892(3)
0.997(3)
1.030(2)
0.998(2)
0.127(3)
0.052(3)
-0.088(5)
0.159(3)
0.176(3)
0.374(4)
-0.041(3)
0.244(3)
-0.013(3)
-0.183(2)
B iso (A2)
10(1)
3.1(4)
3.1(4)
2.6(3)
4.3(5)
3.1(4)
5.8(6)
4.5(5)
5.5(5)
3.7(4)
6.4(7)
5.4(5)
5.6(6)
4.2(5)
5.7(6)
4.9(5)
4.8(5)
3.9(4)
3.5(4)
4.5(5)
5.5(6)
11(1)
6.8(7)
A p p e n d ix
D
CRYSTALLOGRAPHIC DATA FOR
(2R,6R)-6-CARBOXYLATOMETHYL2-HYDROXYMETHYL-2,4,4TRIMETHYLMORPHOLINIUM
C i0H i9NO4.2 H 2O, Mr = 253.3, orthorhombic,
a = 7.8402(7), b =
A3, Z
= 4, Dx = 1.316 g cm '3 a t 292
12.5209(6), c = 13.0286(7) A , V = 1279.0(3)
K, X (Cu K a ) = 1.54184
A,
|i
= 8.7 cm '1, F(000) = 552, 2597 unique d ata
m easured, final R = 0.0334 for 2435 reflections w ith I > 3a(I).
113
114
T a b le D .l. Coordinates and isotropic therm al param eters
fittl=(87C2/ 3 ) £ £ U iia ; a ; a , a i
‘
Atom
01
02
03
04
N
Cl
C2
C3
C4
C5
C6
C7
C8
C9
CIO
01W
02W
X
0.3308(1)
0.1842(2)
0.3618(2)
0.6578(1)
0.1162(2)
0.0530(2)
0.1918(2)
0.4099(2)
0.2752(2)
0.1424(2)
-0.0186(2)
0.1185(2)
0.2329(2)
0.5197(2)
0.5232(2)
0.6750(2)
0.8622(2)
j
y
0.55093(7)
0.7990(1)
0.79070(9)
0.46173(9)
0.46807(9)
0.5417(1)
0.6138(1)
0.4834(1)
0.4118(1)
0.5273(1)
0.3848(1)
0.6755(1)
0.7618(1)
0.5478(1)
0.4066(1)
0.6867(1)
0.7462(1)
z
0.59485(7)
0.77761(9)
0.6447(1)
0.62969(9)
0.43284(9)
0.5158(1)
0.5571(1)
0.5193(1)
0.4696(1)
0.3338(1)
0.4143(2)
0.6469(1)
0.6923(1)
0.4445(1)
0.5803(1)
0.6705(1)
0.8474(1)
B eq (A2)
2.04(2)
4.10(2)
3.78(2)
3.35(2)
2.45(2)
2.35(2)
1.94(2)
2.06(2)
2.36(2)
3.46(3)
4.03(3)
2.18(2)
2.45(2)
2.90(3)
2.45(2)
4.46(3)
5.35(3)
115
T a b le D.2. Coordinates and isotropic therm al p aram eters
for hydrogen atom s
Atom
H 40
H la
H lb
H2
H 4a
H4b
H5a
H5b
H5c
H6 a
H6 b
H6 c
H 7a
H7b
H 9a
H9b
H9c
H lO a
HlOb
H1W1
H2W1
H1W2
H2W2
X
0.705(4)
0.014(2)
-0.042(2)
0.226(2)
0.314(3)
0.240(2)
0.215(3)
0.170(3)
0.028(3)
-0.038(3)
-0.115(3)
0.023(3)
0.092(2)
0.016(2)
0.615(3)
0.557(3)
0.455(3)
0.454(2)
0.561(3)
0.596(4)
0.677(4)
0.949(4)
0.820(4)
y
z
0.414(2)
0.493(1)
0.584(1)
0.659(2)
0.571(1)
0.485(1)
0.503(1)
0.412(1)
0.522(1)
0.346(1)
0.283(2)
0.312(2)
0.660(1)
0.374(1)
0.357(1)
0.587(2)
0.475(2)
0.555(2)
0.344(2)
0.425(2)
0.342(2)
0.628(1)
0.710(1)
0.584(2)
0.499(1)
0.610(1)
0.367(1)
0.353(1)
0.723(2)
0.620(2)
0.781(2)
0.734(2)
0.487(1)
0.389(2)
0.360(2)
0.700(1)
0.624(1)
0.476(2)
0.394(2)
0.405(1)
0.632(1)
0.535(1)
0.663(2)
0.659(2)
0.820(2)
0.791(2)
Siso (A2)
.0 ( 6 )
2.8(3))
2.4(3)
2.3(3)
3.3(4)
2.9(3)
4.2(4)
4.9(5)
6 . 1 (6 )
4.4(4)
5.8(6)
5.6(5)
2.4(3)
3.2(4)
5.5(5)
5.2(5)
4.7(5)
3.0(4)
6
3.6(4)
6.9(7)
7.4(7)
6.3(6)
8.7(8)
A p p e n d ix E
CRYSTALLOGRAPHIC DATA FOR
METHYL ( 2 S , 6 S # R , 6 R ) - 2[6-(2-CYANOETHYL)-4,6DIMETHYLMORPHOLINYL]ACETATE
C 1 2 H 2 0 N 2 O 3 , Mr = 240.3, triclinic, PI, a = 5.6057(4),
14.1151(10)
=
2
A, a
= 74.430(7), p = 88.908(6),
7
b
= 9.1737(7),
c
=
= 79.302(6)°, V = 686.7(1) A3, Z
, D x = 1.162 g cm ' 3 a t 297 K, X (Mo Ka ) = 0.71073
A,
|i
=
0 .8
c m '1,
F(000) = 260.0, 2409 unique d ata m easured, final R = 0.040 for 1850
reflections w ith I > 2a(I).
116
117
T a b le E .l. Coordinates and isotropic therm al p aram eters
»
Atom
0 1
0 2
03
N1
N2
Cl
C2
C3
C4
C5
C6
C7
C8
C9
CIO
C ll
C12
X
0.1478(2)
0.0572(2)
0.4592(2)
-0.1319(3)
-0.6392(4)
0.0433(3)
0.0703(3)
-0.0015(3)
-0.0398(3)
-0.1843(4)
0.2589(3)
0.2421(3)
0.4578(4)
0.1474(4)
-0.2458(3)
-0.2261(3)
-0.4585(4)
j
y
0.3437(1)
0.6770(1)
0.6267(1)
0.1366(1)
0.7041(2)
0.1943(2)
0.3509(2)
0.2750(2)
0.1219(2)
-0.0096(2)
0.4188(2)
0.5856(2)
0.7879(2)
0.2508(2)
0.3815(2)
0.5475(2)
0.6373(2)
z
0.77508(6)
0.5993(1)
0.59987(8)
0.7412(1)
0.8732(1)
0.6707(1)
0.6780(1)
0.8525(1)
0.8395(1)
0.7321(2)
0.6118(1)
0.6039(1)
0.5869(2)
0.9463(1)
0.8519(1)
0.8417(1)
0.8589(1)
B eq (A2)
3.84(2)
6.85(4)
5.33(3)
5.00(3)
8.44(5)
4.97(4)
3.82(3)
4.04(4)
5.19(4)
7.71(6)
4.51(4)
4.34(4)
6.75(5)
5.76(5)
4.33(4)
5.43(4)
5.78(5)
118
T a b le E.2. Coordinates and isotropic therm al param eters
for hydrogen atom s
Atom
X
H la
H lb
H2
-0.007(3)
0.201(3)
-0.087(2)
H 4a
H4b
H 5a
0.118(3)
-0.151(3)
-0.301(4)
-0.242(4)
-0.036(3)
0.229(3)
0.422(3)
0.386(4)
0.369(4)
0.615(4)
H5b
H5c
H 6a
H6b
H 8a
H8b
H8c
H 9a
H9b
H9c
H lO a
HlOb
0.053(3)
0.299(3)
0.187(3)
-0.322(3)
-0.346(2)
Hlla
H I lb
-0.176(3)
-0.118(3)
y
0.200(2)
0.124(2)
0.420(1)
0.046(2)
0.081(2)
-0.046(2)
0.006(2)
-0.093(2)
0.414(2)
0.361(2)
0.819(2)
0.855(2)
0.802(2)
0.214(2)
0.173(2)
0.354(2)
0.340(2)
0.381(1)
0.597(2)
0.554(2)
z
0.606(1)
0.683(1)
0.6595(9)
0.854(1)
0.889(1)
0.783(1)
0.663(1)
0.748(1)
0.547(1)
0.636(1)
0.644(1)
0.525(1)
0.587(2)
1.002(1)
0.945(1)
0.949(1)
0.913(1)
0.7991(9)
0.776(1)
0.890(1)
Biso (A2)
5.5(4)
5.9(4)
4.2(3)
6.6(4)
6.5(4)
10.2(6)
10.3(6)
8.9(5)
5.4(4)
5.9(4)
10.3(6)
10.8(6)
11.3(6)
7.7(5)
7.9(5)
7.9(5)
6.0(4)
4.1(3)
8.7(5)
7.2(4)
VITA
Guobin S un was born in H arbin, P. R. C hina on F ebruary 25, 1961.
He completed his secondary education in China, grad u atin g in 1979. He
received a Bachelor of Science Degree in C hem istry in Ju ly of 1983 and a
M aster's Degree in C hem istry in Ju ly of 1986 a t Peking U niversity, Beijing,
China.
He th e n worked as an assista n t professor a t Beijing Norm al
U niversity, Beijing, China, teaching analytical chem istry.
In J a n u a ry of
1990, he came to Louisiana S tate U niversity in B aton Rouge, where he is
currently a candidate for th e degree of Doctor of Philosophy in the
D epartm ent of Chem istry.
119
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Guobin Su n
Major Field: C h e m is try
Title of Dissertation:
R eactio n -in term ed iate A nalogues:
D esign, S yntheses, and
Inhibitory S tu d ie s with C a rn itin e A c e ty ltra n s fe ra s e
Approved:
/iff!
Major
0
_
Professor(/knd Chairman
^ --
y~z> cx J )_______
Dean of the GraduatevSchool
EXAMINING COMMITTEE:
Date of Examination:
June 20, 1994