The synthesizing of several polyethylene glycols having crown ethers attached is reported. This work led to the identification of three new polymer types which promise to be more effective at selectively binding specific cell types. Work was completed on identification of chemical properties of the new polymer crowns and on development of new techniques for determination of polymer-phase composition

Harris, J. M.

English

NASA Technical Reports Server

THE UNIVERSITY OF ALABAMA IN HUNTSVILLE 
D ~ p a r t m e n t  of  Chemistry 
Huntsv i  1 l e ,  Alabama 35899 
NEW POLYMERS FOR PHASE PARTITIONING 
(Septev j - r  1 ,  1980 t o  August 31, 1981) 
ANNUAL REPORT 
Cont rac t  NAS8-33978 
J. M i l t o n  H a r r i s  
P r i n c i p a l  I n v e s t i g a t o r  
(NASA-CR-lblB70) NEY P C L Y d E K S  FOR PHASE N81-3329 1 
PARTlTIONING Annual b e p o r t ,  1 Scp, 1980 - 
3 1  dug. 1Ydl (Alabdmd U n l v .  IR nuutsville.) 
7 p HC Ad2/HF A 0 1  CSCL 07C Uncl as 
t i 3 / 1 7  L7071 
Prepared f o r  
George C.  Marsha l l  Space F l i g h t  Center 
Na t iona l  Aeronaut ics  and Space A d m i n i s t r a t i o n  
Marshal 1 Space F l  i g h t  Center, Alabama 
September 16, 1981 : 
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https://ntrs.nasa.gov/search.jsp?R=19810024748 2020-03-21T11:17:50+00:00Z
NEW POLYMERS FOR PHASE PARTITIONING - ANNUAL REPORT 
ABSTRACT 
During the past year we have achieved our goal of synthesizing severa l  
polyethylene glycols having crown e thers  attached. Testing of these polymers 
i n  red blood c e l l  separations will begin soon. This preliminary work has 
led t o  the ident i f ica t ion  of three  new polymer types which promise t o  be 
more effect ive a t  se lec t ive ly  binding s p e c i f i c  c e l l  types; these polymers 
are  being synthesized. Work has been completed on iden t i f i ca t ion  of chemical 
properties of the new polymer-crowns and on development of new techniques 
fo r  determination of polymer-phase composition. Examination of the  I t o  de- 
vices fo r  rapid phase-partitioning s tudies  is w e l l  advanced; fu r the r  work 
is t h i s  area awaits repai r  of the  ins t rumwt a t  Marshall. 
- 
NEW POLYMERS FOR PHASE PARTITIONING - ANNUAL REPORT 
I. WORK PERFORMED 
A. Polymer Synthesis 
During the  pas t  year  w e  have achieved ou r  goa l  of synthesizing s e v e r a l  
polyethylene glycols  having crown e the r s  a t tached.  The compounds were prepared 
by.:use of react ions 1 and 2 o r  3 and 4, where TsCl is  2-toluenesulfonyl ch lo r ide ,  
the  crown alcohol is e i t h e r  hydroxymethyl-16-crown-5 o r  hydroxyme thyl-19-crown-6, 
the crown amine is diaza-18-crown-6, and PEG i s  polyethylene glycol.  
NaH 
PEG-OH + TsCl-> PEGOTS 
benzene 
NaH 
PEGOTs + crown-OH --> PEG-crown 
benzene 
PEGOH + Cr03C1(C6H5N) CH2C12 > PEG-CHO 
PEGCHO + crown amine + NaBH3CN -> PEG-crown ( 4) 
The crown alcohols  were synthesized by reac t ion  5. A l l  o ther  compounds 
were purchased. 
A great  dea l  of experimentation was required t o  determine the  proper  
conditions fo r  each of t he se  reac t ions .  With t h e  exception of r eac t ion  5 
none of these reac t ions  had been done before. I n  t he  case of reac t ion  5 t h e  
published report  neglected t o  give s u f f i c i e n t  d e t a i l s  t o  permit ready comple- 
t i o n  of t h i s  synthesis ,  So, I n  e f f e c t ,  a l l  of t h e  chemistry was new. 
The polyethylene g lycols  used were obtained from Union Carbide and 
were of  molecular weights 3400 glmol and 6800 g/mol. Determination of polymer 
y i e ld s  was done by chemical ana lys i s  and by molecular-weight determination 
by size-exclusion chromatography. This  use of size-exclusion chromatography 
was new and was made poss ib le  by Var i an t s  development of the necessary chroma- 
tography columns f o r  water-soluble polymers. 
B. Chemical Proper t ies  of t he  New Polymers 
Before beginning phase p a r t i t i o n i n g  of c e l l s  it i s  important t o  de te r -  
mine some of t he  chemical p rope r t i e s  of  t h e  new polymers. The prime property 
i n  which we a r e  i n t e r e s t e d  is the  a b i l i t y  of these  mater ia l s  t o  bind metals. 
This property can be examined by studying t h e  a b i l i t y  of t h e  polymers t o  a c t  
as phase t r a n s f e r  ca t a ly s t s .  A phase t r a n s f e r  c a t a l y s t  a c t s  by removing a 
metal ca t ion  and its accompanying anion from water i n t o  an immiscible organic  
l i q u i d  layer .  Once the anion is i n  t he  o rgan ic  l i q u i d  i t  i s  very poorly 
so lva ted ,  and a s  a consequence much more r eac t ive .  We have determined t h e  
a b i l i t # e s  of our PEG-crowns and PEG i t s e l f  t o  ca ta lyze  t h e  displacement of 
ch lor ide  from benzyl ch lor ide  by potassium a c e t a t e  i n  a c e t o n i t r i l e .  The 
a c e t a t e  ion normally has only very s l i g h t  s o l u b i l i t y  i n  a c e t o n i t r i l e ,  s o  t h i s  
reac t ion  i s  normally very slow (a  few percent  reac t ion  a f t e r  24 hours). 
We found t h a t  the  crown polymers a r e  very  e f f e c t i v e  phase t r a n s f e r  agents. 
More i n t e r e s t i n g  was the  observat ion t h a t  t h e  PEG'S a r e  a l s o  e f f e c t i v e  agents ,  
although not  q u i t e  a s  powerful as t h e  crown-polymers. This f a c t  is  very impor- 
t a n t  i n  guiding our fu ture  c e l l  p a r t i t i o n i n g  experiments, s i n c e  i t  ind i ca t e s  
t h a t  t he  metal-complexing a b i l i t y  of  t h e  crowns w i l l  be reduced because of t h e  
competition from polymer chains. 
- 
C. Techniques For Determination of Polymer-Phase Composition 
We have done preliminary experiments wi th  the  conventional dextran-PEG 
phase system f o r  c e l l  pu r i f i ca t i on  ( see  next s ec t i on ) .  I n  t h e  course of 
t h i s  work we discovered tha t  t he  conventional techniques f o r  analyzing t h e  
composition of t he  phase systems was woefully de f i c i en t .  The problem is 
three-fold.  F i r s t ,  i t  is very d i f f i c u l t  t o  d ry  dextran,  so t h a t  s tock so l -  
u t i ons  a r e  made from mater ia l  containing an unknown amount of  water. Second, 
the usual  technique f o r  measuring t h e  amount of polymers i n  mixtures of PEG 
and dextran is  t o  determine the dextran by polar imetry,  freeze-dry the solu- 
t i o n ,  and determine the PEG by weight d i f fe rence .  This technique j s  tedious 
and inaccurate .  Third,  and most problematic,  phase p a r t i t i o n i n g  r e s u l t s  
can vary dramatical ly  fo r  phase systems which appear t o  have t h e  same com- 
pos i t ion .  This r e s u l t s ,  we bel ieve,  because of e i t h e r  t r ace  amounts of low- 
molecular-weight impuri t ies  o r  va r i a t i ons  i n  molecular weight of t h e  polymers. 
The three  problems have been solved by app l i ca t i on  of t he  techniques 
of refractometry,  polarimetry,  size-exclusion chromatography (on the  high 
performance l i q u i d  chromatograph), and gas chromatography. Refractometry 
provides a very s e n s i t i v e  measure of t h e  concentrat ions of PEG o r  dextran i n  
a pure aqueous so lu t ion .  A s  was previously known, dextran concentration can 
be determined by polarimetry. To determine the  concentrat ions of a PEG 
dextran mixture i t  is necessary only t o  determine the  dextran concentration 
by polarimetry,  sub t r ac t  the  dextran cont r ibu t ion  t o  t h e  so lu t ion  r e f r a c t i v e  
index, and ca l cu l a t e  the  PEG concentration from the  r e s u l t i n g  calculated re- 
fractive index. This technique i s  very usefu l  and e a s i l y  appl ied  on a rout ine 
bas i s .  
An a l t e r n a t i v e  technique, .which i s  a c t u a l l y  more revea l ing ,  is size-  
exclusion chromatography. This technique not only gives  the concentrat ion 
of PEG and dextran,  but  i t  a l s o  provides a molecular weight p r o f i l e  of t h e  
polymers; i t  i s  t h i s  l a s t  p roper ty  t h a t  c o n t r i b u t e s  t o  v a r i a t i o n s  i n  phase 
p a r t i t i o n i n g  which occur  d e s p i t e  constancy of phase composition ( t h e  t h i r d  
problem mentioned above). Th i s  technique is  a l i t t l e  more complicated t o  
apply  than refractometry-polarimetry, and t h u s  is  n o t  a s  u s e f u l  on a r o u t i n e  
b a s i s .  F i n a l l y ,  we have found gas chromatography t o  be a u s e f u l  technique 
f o r  d e t e c t i n g  t h e  presence of v o l a t i l e  i m p u r i t i e s  i n  the  polymers. These 
i m p u r i t i e s  can a l s o  c o n t r i b u t e  t o  v a r i a t i o n  i n  phrse  p a r t i t i o n i n g  r e s u l t s .  
A s  a r e s u l t  of t h i s  work, we can now c a r e f u l l y  monitor t h e  composition 
and p u r i t y  of our polymer s o l u t i o n s .  This should  a i d  g r e a t l y  i n  t h e  per- 
formance of the  second p o r t i o n  of our  work, t h e  determinat ion o f  t h e  u t i l i t y  
of t h e  new polymers f o r  phase p a r t i t i o n i n g .  
D. Examination of t h e  I t o  Device 
D r .  Y. I t o  (of NIH) has  developed a new dev ice  pe rmi t t ing  rap id  mul t ip le  
phase p a r t i t i o n i n g  experiments.  D r .  I t o  loaned two of t h e s e  devices  t o  r. 
Robert Synder 's  l abora to ry .  We decided t o  spend some time experimenting 
wi th  t h e  new devices  t o  s e e  i f  they would f a c i l i t a t e  t h e  s tudy  of our new 
polymers. Af te r  much exper imentat ion wi th  t h e  un tes ted  machinery we were 
a b l e  t o  reproduce some e a r l y  experiments o f  I t o ' s  and t o  ga in  some under- 
s t a n d i n g  o f  t h e  o p e r a t i o n a l  p r i n c i p l e s  of t h e  ins t ruments .  One device ,  t h e  
nonsynchronous c o i l  p l a n e t  c e n t r i f u g e ,  was deemed s u i t a b l e  f o r  c e l l  separa t ions .  
Unfor tunate ly ,  t h e  ins t rument  was n o t  very durab le ,  and w i l l  n o t  func t ion  
a t  p r e s e n t  because o f  s e v e r a l  expensive mechanical f a i l u r e s  (e.g. ,  t h e  
d r i v e  motor). We do now have s u f f i c i e n t  exper ience  on t h e  dev ice  t o  permit  
i t s  a p p l i c a t i o n  t o  t e s t i n g  of our new polymers should t h e  instrument be  re-  
pa i red .  
11. WORK TO BE PERFORMED 
The major goal of our work is  t o  develop new polymers f o r  use i n  phase- 
par t i t ioning c e l l  pur i f ica t ions .  Now that  we have some of these polymer- 
crowns i n  hand, our next task  w i l l  be t o  examine the polymers i n  our 
proposed t e s t  system, tbp separation of red blood ce l l s .  
In the course of t h i s  work i t  has become obvious t h a t  we now have the 
chemical techniques avai lable  t o  prepare three new polymers having even 
more likelyhood than the  PEGcrams of introducing s e l e c t i v i t y  i n t o  the  
part i t ioning process; we a l so  plan t o  synthesize these polymers. The 
f i r s t  polymer i s  t h a t  resul t ing  from binding antibodies t o  PEG. The anti-  
bodies w i l l  bind s p e c i f i c  groups on c e l l  surfaces. The econd polymer is 
made by binding cyclodextrins t o  PEG. The cyclodextrins a r e  s imi la r  t o  the  
crown e thers ,  but  other  experiments indica te  tha t  they are more l i k e l y  tc ;  
bind the negative binding groups on the  c e l l  surface. The t h i r d  polymer 
is  made by at taching long hydrocarbon t a i l s  (e.g., eighteen carbons) t o  
the PEG by an e the r  linkage. Experiments i n  Don Brook's laboratory have 
shown that  these tails can be very se lec t ive  f o r  binding c e r t a i n  c e l l  types. 
Previously, however, the t a i l s  have been attached by e s t e r  linkages and have 
proven t o  be unstable. We believe tha t  attachment v i a  an e t h e r  linkage w i l l  
give a s t ab le  and thus much more useful  material.  These three polymer types 
w i l l  be examined i n  red blood c e l l  separations. 
111. CURRENT PROBLEMS 
A t  present the  only problem which might impede the  performance of our 
work is the  a v a i l a b i l i t y  of the  I t o  device. We can conduct our experiments 
. 
without t h i s  instrument, but its ava i l ab i l i ty  could speed our t e s t ing  process. 
FINANCIAL STATUS REPORT 
Contract N o .  NAS8-33978 
Period: 9/1/80 - 8/31/81 
1 . Contract Value 
2. 1st increment a l l o t t e d  
3. Expenditures to date 
4. Estimated Funds to completion 
5.  Anticipated over/under run 
6. Changes a~thorized but not finalized 
7. Changes under consideration but not authorized 
*$47,716 s t i  1 1  to  be a1 lo t ted  


New polymers for phase partitioning

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