Transcript Hongyi Cai

Oligomerization of b-Cleft Cross-Linked Human Hemoglobin: Synthesis, Intramolecular
Cross-linking, and Intermolecular Coupling Studies with Polyfunctional Organic Reagents
Hongyi Cai, Timothy A. Roach and Ramachandra S. Hosmane.
Laboratory for Drug Design and Synthesis, Department of Chemistry & Biochemistry, University of Maryland, Baltimore County ,
1000 Hilltop Circle, Baltimore, MD 21250.
Abstract:
Stroma-free
hemoglobin
Lys82 b1
Current efforts to develop blood substitutes based on cell-free hemoglobin
are directed toward (a) tuning its oxygen affinity to afford adequate oxygen
delivery from lung to tissues via covalent cross-linking with an appropriate
reagent that mimics the hemoglobin's natural allosteric modifier 2,3diphosphoglycerate (DPG), and (b) increasing the steric bulk of the crosslinked hemoglobin to allow its retention in circulation for prolonged
periods of time as well as to prevent its facile seeping through the
endothelium and the subsequent interaction with nitric oxide, which
results in vasoconstriction, and hence, the elevated blood pressure. As
part of a program to address the current problems facing the blood
substitute research, we report here the results of our studies on sequential
intramolecular cross-linking and intermolecular coupling of human
hemoglobin, employing a variety of polyfunctional organic reagents.
NH2
Lys144 b2
H2N
+
HO
O
O
O
O
P
OH
BCCEP
Or
OH
O
O
O
P
OH
O
O
HO P O
OH
O
Synthesis III: Synthesis of Bis-Mal-PEG2000
O
O
O
O
BPPCEP
O
O
O P OH
OH
NH2
Lys82 b1
O
acetic anhydride
O
+
O
P
O
N
H
4-aminobenzoic acid
Lys144 b2
N
H
SH
P
Cys93 b2
HS
O
O
H
N
Ph
O -CH2CH2-(OCH 2CH2)45- O
C
N
H
N
O
N
DPPA
N
N
dry toluene
N
N
O
S
O -CH 2CH2-(OCH2CH2)45- O
N
Figture 4. (A) C4 reverse phase chromatogram of stroma-free hemoglobin
Monitored at 214nm. (B) C4 reverse phase chromatogram of stroma-free
hemoglobin, modified by Bis-Mal-PEG2000, Monitored at 214nm.
O
p-maleimidobenzoyl azide
C
N
H
p-Maleimidophenyl isocyanate
N
O
O
S
Cys93 b2
HO-CH2CH2-(OCH2CH2)45-OH
bb-Crosslinked hemoglobin at Cys
O
PEG-2000
toluene/30-40 C, 3h
Introduction:
N
O
O
NH C O-CH2CH2-(OCH2CH2)45- O C N
H
O
In a summary, we have demonstrated the intramolecular
cross-linking (32KDa) of Hb with these reagents.
Although the expected 64 kDa Hb dimer was not seen,
partial intermolecular cross-linking (48KDa) with the
above reagents was observed. It is possible that Hb
changes its conformation after reacting with an reagent,
causing the activity site to another cross-linking reagent
to be hindered. We are currently altering the reaction
conditions and purifying the intermediate products to
optimize reaction efficiency.
N
O
Bis(maleidophenyl)-PEG2000
Results and Discussion
Figure 2. Experimental Procedures
Scheme I: Synthesis of BCCEP
O
O
OH +
HO
t-Butanol
N C N
O
Oxalic acid
4-Hydroxy benzoic acid
1,3-dicyclohexylcarbodiimide(DCC)
O
O
KO
H3C
O
/ THF
4-t-butoxycarbonylphenyl acrylate
O
O
O
O
(B)
(C)
I would like to express my great appreciation to my
advisor, Dr. Ramachandra Hosmane, who has
supported me throughout this work. In addition, I want
to thank Dr. Tim Roach for his instruction and
encouragement on this project. Finally, I would like to
thank my colleagues and friends for all of their helpful
suggestions.
O
TFA
P
O
O
OH
CH2Cl2 / reflux
Bis[2-(4-t-butoxycarbonyl)phenoxycarbonylethyl]phosphinic acid
O
O
HO
O
O
O
O
P
O
Acknowledgements:
OTMS
NH4H2PO2 / CH2Cl2
O
O
(A)
4-Hydroxy-t-butylbenzoate
O
O
OH
The structures of three reagents were as determined by 1H NMR
(Figure 2), Mass Spectroscopy and IR. The extent of the crosslinking globin chains was determined by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3) and C4
reverse phase High performance liquid chromatography (HPLC)
(Figure 4).
NTMS
Cl
OH
OH
Bis[2-(4-carbonxyphenoxy)carbonylethyl]phosphinic acid (BCCEP)
References:
Figture 2. NMR analysis of BCCEP(A), BPPCEP(B) and Bis-Mal-PEG2000(C).
Scheme II: Synthesis of BPPCEP
O
HO
O
O
H
O
O
Experimental Procedures
O
O
O
P
OH
O
CH2Cl2, TFA, Reflux
O
O
Bis[2-(4-tert-butoxyphenoxy)carbonylethyl] Phosphinic Acid
O
HO
O
O
O
P
O
O
2) H2O2, -76'C
P
OH
O
O
O
P
OH
48KDa
32KDa
16KDa
OH
Bis[2-(4-hydroxyphenoxy)carbonyletyl] Phosphinic Acid
HO
(B)
1)di-tert-butyl -diisopropyl
-phosphoramidite/1H-tertazole, DMF
OH
O
(A)
1. R. S. Hosmane, S. P. Peri, V. S. Bhadti and V. W. Macdonald. Bis[2-(4carboxyphenoxy) carbonylethyl] phosphinic Acid (BCCEP): A Nevel Affinity Reagent
for the β-Cleft Modification of Human Hemoglobin. Bioorg. Med. Chem., 1998, 6,
767-783.
4-t-butoxypehnyl acrylate
O
O
H
TMSCl, Et3N, CH2Cl2
4-(t-butoxy)phenol
1
P
ONH4
Cl
KOt-Bu
Two cross-linking reagents previously designed in our lab - BCCEPE and
BPPCEP 2, reacting with Lys82 and Lys144 of β chains were synthesized in
schemes I and II, respectively. Bis-Mal-PEG2000 was reported to cross-link
at Cys-93 of β subunits 3, and was synthesized using scheme III 4. Then
Hemoglobin was reacted with BCCEPE or BPPCEP and Bis-Mal-PEG2000 in
a series of reaction conditions.
N
O
O
C
N
H
C
reflux
PH=6.5-7.5
O
O
O
O
O
Attempts to develop a red blood cell (RBC) substitute in transfusion date
back well over half a century. Hemoglobin (Hb), the natural oxygen carrier
inside the RBC has been the preferred choice for such a substitute.
Unfortunately, when Hb is outside of the RBC, its affinity to oxygen
increases to an extent that may impair oxygen delivery from lungs to
tissues. Furthermore, it suffers from short circulation times in the blood
stream due to its breakdown from a large tetrameric protein into two
smaller dimeric units, α1β1 and α2β2. These drawbacks are due to the
loss of the natural Hb allosteric effector, 2,3-Bisphosphoglycerate (BPG).
BPG aligns itself between the two β subunits of the Hb tetramer, and is
surrounded by several positively charged amino acid residues residing on
the β subunits, thus forming an anionic sink that is referred to the β-cleft
or the BPG pocket. Therefore, the covalent cross-linking of Hb subunits
with a BPG mimic, preferably in the β-cleft site, is anticipated to alleviate
these drawbacks of cell-free Hb. We designed and synthesized several
organic reagents (BCCEP, BPPCEP & Bis-Mal-PEG2000) for hemoglobin
cross-linking studies. These reagents have been shown to modify Hb
between different amino-acid residues in β chains. We propose to obtain
intramolecular and intermolecular cross-linkedβchains by sequential
reactions with these reagents.
O
O
N
Bis-Mal-PEG2000
O
N
dry toluene /triethylamine
O
O
C
p-Maleimidobenzoic acid
O
+
Cys93 b1
N
HO
Maleic anhydride
Ph
Stroma-free
hemoglobin
N
(B)
O
O
bb-Crosslinked hemoglobin at Lys
N
O
O
Sodium acetate
O
Cys93 b1
O
O
HO
(A)
O
O
O
O
O P
OH
OH
Bis[2-(4-phosphonooxyphenoxy)carbonylethyl] Phosphinc Acid(BPPCEP)
3) CH2Cl2, TFA
Figture 3. SDS-PAGE analysis of the cross-linked Hb. (A) Hb reacted with
BCCEP (HY-20) and Bis-Mal-PEG2000 (HY-14), (B) Hb reacted with
BPPCEP (HY-8) and Bis-Mal-PEG2000 (HY-14). Lane 1, MW standards;
XL,cross-linking
48KDa
32KDa
16KDa
2. T. A. Roach, V. W. Macdonald, and R. S. Hosmane. A Novel Site-Directed Affinity
Reagent for Cross-Linking Human Hemoglobin: Bis[2-(4-phosphonooxy
phenoxy)carbonylethyl]phosphinic Acid (BPPCEP). J. Med. Chem., 2004, 47, 58475859.
3. B. N. Manjula, A. Malavalli, P. K. Smith, N. Chan, A. Arnone, J. M. Friedman and A. S.
Acharya. Cys-93- -Sucinimidophenyl Polyethylene Glycol 2000 Hemoglobin A
Intramolecular Cross-bridging of Hemoglobin Outside The Central Cavity. J. Biol.
Chem, 2000, 275, 5527-5534.
4. M. E. Annunziato, U. S. Patel, M. Ranade, and P. S. Palumbo. p-Maleimidophenyl
Isocyanate: A Novel Heterobifunctional Linker for Hydroxyl to Thiol Coupling.
Bioconjugate Chem. 1993, 4, 212-218.