biomimetic aminoacylation of rna and other 1,2-diols · alteration by synthetic incorporation 3...
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Biomimetic aminoacylation of RNA and other 1,2-diols
1
Ronald KlugerDeparment of Chemistry, University of Toronto
Molecular diversity in proteins Native
Sequence as specified by genetic code
Altered By chemical modification
Side chains modified By mutagenesis
Side chains intact
2
Alteration by synthetic incorporation
3
From: S. L. Monahan (2004) Site-specific incorporation of unnatural amino acids into receptors expressed in mammalian cells.Dissertation (Ph.D.), California Institute of Technology.
Adding non-coded amino acids
4
Exonuclease to remove CpA from 3’ terminus
Synthesize aminoacyl-tRNA
Ligate to restore 3’-terminus
Permits misacylation
4
Hecht et al. Biochemistry 1987 26, 3197.Schultz et al. Science, 1989 244, 182
55
Ligation to RNA
Multiple steps
N
NH2
ON
O
OPOO
O
O-
N
NN
N
NH2
O
OHOH
PO
OO
N
NH2
ON
O
OPOO
O
O-
N
NN
N
NH2
O
OHO
PO
OO
O
N
NH2
ON
O
OPOO
O
O-
N
NN
N
NH2
O
OHO
PO
OtRNApheOH
O
pCpA aminoacyl pCpA Misacylated tRNA
T4 RNA Ligase
O
OHO
tRNA -Phe
O
OCCNNHNVOC
R
NHNVOCR
1. deprotect
RNH2
A general and efficient route for chemical aminoacylation of transfer RNAs P.G. Schultz et al.J. Am. Chem. Soc. 1991,113, 2722. 6-Nitroveratryloxycarbonyl
NVOC
6
Aminoacylation bio-catalysis Bertozzi, Tirrell et al.
Discovery of Aminoacyl-tRNA Synthetase Activity Through Cell Surface Display of Noncanonical Amino Acids. PNAS 2006, 103, 10180.
Szostak et al.Enzymatic aminoacylation of tRNA with unnatural amino acids PNAS 2006 103 4356 “we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties.”
Suga et al.Ribozyme-Catalyzed tRNA Aminoacylation in Aminoacyl tRNA Synthetases, 2007 Michael Ibba, editor
Schultz et al.An efficient system for the evolution of aminoacyl-tRNA synthetase specificity Nature Biotechnol. 2002 20, 1044. “we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein.”
HO OH
O NN
NH2
NN
PO
OO OOP
O
OOPO
HO+
NH2
RO
OHO OH
O NN
NH2
NN
PO
OOO
NH2
RO
tRNA
HO OH
O NN
NH2
NN
PO
OOHO
NH2
RO
O OH
O NN
NH2
NN
PO
O
O
Aminoacyl adenylate
APPLICATIONS AND COMMERCIALIZATION
Next step
7
Ion channels and receptors
8
Nicotine binding to brain receptors requires a strong cation– interaction, Dougherty et al.Nature 2009 458, 534
a, Structures of ACh and nicotine. b, Unnatural amino acids considered here. If not indicated, an a, b, c, or d group is H. Br, bromo group; CN, cyano group.c. The backbone ester strategy for modulating a hydrogen bond.
Probes of nicotine receptor
9
“‘Fuorination’ trend is seen for both ACh and nicotine at TrpB of the α4β2 receptor. This is in contrast to the results at the muscle-type receptor, in which no such trend is seen for nicotine activation”
Reagents “Dissolve unnatural aminoacyl-tRNA with tRNA buffer, and mix
with template DNA and cell-free translation system. The mixture is incubated for 1 hour to synthesize protein containing unnatural amino acid.”
“Full-length protein containing unnatural amino acid can be isolated by purification for C-terminal tag such as His tag.”
http://cosmobiousa.com/131cloverdirect.html
Protein optimization
11
“Ambrx uses an expanded set of amino acids to address the limitations intrinsic to the 20 natural amino acids. Our pioneering protein medicinal chemistry™ drug development process combines the power of medicinal chemistry with recombinant biosynthesis.”
http://www.ambrx.com/wt/page/protein_optimization
Biosuperiors
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“Sutro's biochemical protein synthesis technology allows for the rapid incorporation of a wide variety of nnAAs because the charged tRNA is added as a separate reagent to the scalable biochemical protein synthesis reaction.”
http://www.sutrobio.com/tech/biosuperiors.html
ACYL PHOSPHATE MONOESTERS
Key functionality
13
Biomimetic approach Chemical reactions should be independent of side chain Need to activate (analogue of AMP) Need to gather (analogue of AATRS)
14
UO
OHO
AO
OHO
OP-O
O
H2NO
R
UO
OHO
AO
OHHO
OP-O
O
Berg (1956): acyl adenylates
15
“I decided to synthesize acetyl adenylate chemically. Being a novel compound, I contacted David Lipkin, a specialist in phosphate chemistry, on how to proceed. It's easy, he advised: mix acetyl chloride and the silver salt of adenylic acid, remove the insoluble silver chloride, and collect the mixed anhydride from the fluid. Within a week or so the first ever batch of pure acetyl adenylate was available and I could verify that the enzyme converted it rapidly and quantitatively to ATP in the presence of only PPi and to acetyl-CoA with added CoA. The overall reaction could then be explained as the result of two successive steps.”1
1Berg, P. Acyl adenylates: the synthesis and properties of adenyl acetate. J. Biol. Chem. 1956 222, 1015-1023
From Berg, P. , Moments of discovery: my favorite experiments J. Biol. Chem. 2003, 278, 40417-40424.
ATP + acetate Acetyl adenylate + PPiAcetyl adenylate + CoA Acetyl CoA + AMP
16
Acyl phosphate monoesters
Anionic acylating agents, same as AA-AMP Specificity from shape, charge, functional groups
H3C
O
OP
OO
OCH3H3C
O
OP
OOCH3
OCH3
NaI
H3C
O
H3COP
OOCH3
OCH3Cl
+Na+
MAP
Biochem. and Cell Biol. 1986 64 434-440.
H3C
O
CO
O
H3C O
O
PO
O
OCH3
E E
H2NNHH
H
17
Targeting the DPG site of hemoglobin
2,3-diphosphoglycerate DPG is a penta-anion Anionic agent targets
specific site Acylation targets Lys
O
O
O
O
P
O
O
O
P
O
O
O
VAL 1LYS 82
LYS 82
VAL 1
'++
+
+
H3C
O
OP
OO
O CH3
H2NHb
MAP
J. Biol. Chem., 1989 264, 12344-1235Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins
O
O
O
H3C
O
Aspirin
lys 82
18
Site-directed cross-linkers
Anionic, defined span, reactive toward amines
R
O
ClCl
O+ 2
OP
OOCH3
OCH3R
OO
OP
OOCH3
OCH3O
P
OH3CO
H3CONaI
R
OO
OP
OO
OCH3O
P
OO
H3CO
Na+Na+
R =
R. Kluger, A. S. Grant, S. L. Bearne, M. R. Trachsel J. Org. Chem. 1990 55, 2864-2868.
Acyl pyrophosphates Activated analogues of ATP, ADP, Acetyl phosphate
19
P
O
OO-O-
OP
O
O-
O
H3C
P
OO
OO
OHHO
O
N
N
H2N
N NP
OO
O
P
OO
OO
OHHO
O
N
N
H2N
N NP
OO
O
O
ADP
AcADP
Acyl pyrophosphates: activated analogs of pyrophosphate monoesters permitting new designs for inactivation of targeted enzymesR. Kluger, Z. HuangJ. Am. Chem. Soc., 1991, 1135124
Labels RPP binding site
20J. Am. Chem. Soc., 1991, 113 5124
Kinase interrogation Kozarich, ActivX Biosciences, Inc., A wholly owned subsidiary of
Kyorin Pharmaceutical
21
Biochemistry, 2007, 46 (2), pp 350–358
Acyl-nucleotide probes and methods of their synthesis and use in proteomic analysis US 7,365,178 Campbell et al. April 29, 2008
Advice
22
Aminoacyl adenylates
23Moldave, Castelfranco, and Meister, J. Biol. Chem. 1959 234 841.
2424
Aminoacyl phosphate monoesters
R. Kluger, X. Li, and R. W. Loo, Can. J. Chem. 1996, 74 2395.
2525
Reactivity Anionic leaving group: repelled by anions, attracted by cations React rapidly with amines (useful for protein modification) React slowly with oxygen nucleophiles – not good for tRNA
W. P. Jencks and J. Carriuolo J. Biol. Chem. 1959, 234, 1272, 1280.G. DiSabato and W.P. Jencks, J. Am. Chem. Soc. 1961, 83, 4400.
2626
Lewis acid activation
metal k2, s-1 K1, M-1
Cu(II) 2.6 × 10-2 250 Zn(II) 2.7 × 10-2 141 Mg(II) 1.0 × 10-2 28 Ca(II) 0.9 × 10-2 15
pH 7.0, 25 ° C Modest acceleration
kHOH = 3 x 10-5 s-1t1/2 ~ 400 minutes
J. Am. Chem. Soc. 1997 119 12089.
LANTHANIDE TEMPLATE CATALYSIS
Binding site and Lewis acid
27
2828
Activation by lanthanides Stable and water soluble Hard Lewis acids, high coordination number and
unrestricted geometry
2929
Lanthanides accelerate hydrolysis of BzMP
0.010.0080.0060.0040.0020
4
2
0
[Ln3+], M
1000
kob
s s-1
pH 7, 250C10 mM EPPS
.
.
.
.
.
Metal K1, M-1 k2, s-1 k2 x K1
NdOTf3 2 x 102 4 x 10-3 0.88
EuOTf3 4 x 102 4 x 10-3 1.6
EuCl3 2 x 102 6 x 10-3 1.3
YbOTf3 8 x 102 2 x 10-3 2.3
LaOTf3 3 x 102 1 x 10-3 0.53
MgCl2 - - 8 x 10-6
no metal 2 x 10-7
J. Am. Chem. Soc. 2002 124 3303
CO
O
P
OO
ONa
CH3
3030
Internal addition Reaction between coordinated ligands Entropic advantage
CO
OP O
O
OCH3
La+3
O
OP O
O
OCH3
La+3HO
H2O
H2O
pKa = 9
OH-
CO
OP O
O
OCH3
La+3
OH
C HO
OP O
O
OCH3
+3La
O
J. Am. Chem. Soc. 2004 126 10721.
3131
Refinement: acylation of alcohols
Does this parallel hydrolysis?
3232
BzMP in 50:50 water:methanol
10-3 M La+3, 10-2 M EPPS, pH 8
20181614121086420
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
t=2hr 20min
20181614121086420
0.28
0.24
0.2
0.16
0.12
0.08
0.04
0
time/ min
abs
t=0
Ester
BzCOOH
BzMP
before
after
3333
Reactivity – selective for cis diols in water
krel = 1 for hydrolysis Cis or flexible diols are particularly reactive
No diester products – only one OH reacts
O
O
O
O
krel = 20 krel = 0.08
O
O
krel = 170OH
O
O
OH
krel = 17
O
O
OH
krel = 82, 11
O
O
OH
krel = 46
O
O
OH
krel = 0
O
OOH
krel = 49
3434
Diol reactions implicate chelation Indicates kinetically significant nucleophilic coordination
Results in a low-entropy reaction
O
OP O
O
OCH3
Ln 3+OHHO
H+
O
OP O
O
OCH3
Ln 3+OHO
O
OP O
O
OCH3
Ln3+
OHHO
35
Nonaqueous Analogy: Monoacylation
Catalytic monoacylation of diols (1,2; 1,3; 1,4) dichloromethane
Via bis-bidentate complex
P. A. Clarke et al. Chem. Commun., 2003, 2588 lanthanide (III) salt catalysed monoacylation of symmetrical diols from structural models
Crystal structure of analogue
3636
Possible reactions of adenosine + BzMP
N
NN
N
NH2
O
OHOH
HO
OPO-
OCH3
OO
+
N
NN
N
NH2
O
OHOH
O
O
5' acylation
N
NN
N
NH2
O
OO
HO
O
2' / 3'acylation
La3+, H2O
N
NN
N
HN
O
OHOH
HO
O
exocyclic amine acylationAdenosine
3737
No diol, no reaction
N
NH2
ON
O
H
HH
HH
HO
H
2', 3'-2Dideoxycytidine (DDC)
Neither OH nor NH2 react
2×10 -3 M 2’,3’-DDC, 5× 10-4 M BzMP, 1 ×10-3 M LaCl3, 1× 10-2 M EPPS, pH 8.0, room temperature.
3838
Deoxynucleosides
No acylation with BzMP & La+3
3939
Nucleosides require 1,2-diol
Analogues with modifications at reaction sites Observe: two acylation products or none
2’(3’)-diol required for acylation with La+3
4040
3’5’-cyclic AMP – no reaction
N
NN
N
NH2
O
OHO
HHHH
PO
O-
O-
O5'
3't(min)
0 2 4 6 8 10 12 14 16
abso
rban
ce(2
30 n
m)
0
0.05
0.1
0.15
0.2
0.25
RCOOH
cAMP
BzMP + La+3 etc.
4141
Dinucleotide acylation
Monoacylation product Yield: 53 %
ApC = Terminus of tRNA Used in Hecht, Schultz
processes
1×10 -3 M ApC, 5× 10-4 M BzMP, 2 ×10-3 M LaCl3, 2.4 h.
N
NH2
ON
O
OHH
N
NN
NNH2
O
OHOPOO-
HO
O-
OH
4242
Reaction at 3’-terminal of RNA
Mg++ binds to phosphates
Unique diol –binds La+3
Also, Mg++ will allow La+3 to be released from by-products
La+3
Mg++
Mg++
4343
Aminoacylation of AMP Tetraethylammonium N-t-BOC tyrosine ethyl phosphate (TEP) Esters form (mass spec + HPLC analysis)
4444
HPLC analysis
HydrolysisTEP
Esters
AMP
Time0 20 40 60
A
0
0.2
0.4
0.6 Reaction
Control
Tyrosine ethyl phosphate = TEP
45
Analysis of reaction with RNA HPLC – not enough change to cause separation in
macromolecule MS requires homogeneous sample, does not identify sites Alternative: introduce an unnatural unique signal in the
aminoacyl group 19F NMR
relatively sensitive fluorinated amino acids are available
Tzvetkova, S.; Kluger, R. J. Am. Chem. Soc. 2007, 127 15848
Dhiman, R.S.; Kluger, R Org. Biomol. Chem., 2010, 8, 2006
46
19F NMR – aminoacylation of cytidine
FNHBoc
O
OP
O
OO-
BocFPEP19F NMR: - 117.2 ppm
Cytidine
Time, min
0 20 40
AU (2
63 n
m)
00.20.40.60.8
11.21.41.61.8
22.2
Esters(m/z 509.2)
BocFPhe(19 F NMR-117.8 ppm)
BocFPEP
Esters
19F NMR (282 MHz)Reaction mixture quenched with EDTA at 10 sec (LaCl3)
Cytidine aminoacylation 3 minutes
NMR
HPLC
HO
HO OH
O N
ON
NH2
cytidineHO
O OH
O N
ON
NH2
OR
NHBOC
+
REACTIONS WITH RNA
47
4848
Reaction with RNA mixture
After one hour – without purification
19F NMR (376 MHz, D2O)
After purification and dilution
19F peaks are incorporated into RNA
RNA mixture, BocFPEP, LaCl3, MgCl2, pH 8; 1 hr
G-25
RNA and BocFPheRNA
Esters
4949
Is reaction at 3’-terminus of tRNA?
Oxidize RNA with NaIO4 Convert diol to dialdehyde
N
NN
N
NH2
O
OHOH
HHHH
OPOO-
O
N
NN
N
NH2
O
OOHH
HH
OPOO-
ONaIO4
o-tRNA
Method: Nucleic Acids Res. 1996, 24, 4535
5050
Periodate oxidation prevents reaction
After one hour After purification –hydrolysis product remains
No F in o-tRNAEstablishes that incorporation is at 3’ terminus
19F-NMR
G-25
N
NN
N
NH2
O
OO
OPO
O-
O
No esters
5151
DNS- aminoacyl ethyl phosphates
N
SO OHN
O OP
O O
O-
N
SO ONH
NHBocO
O
P-O OO
HNO
O
N
S OOPO-
OO
NHO
O
S
SO O
N
PO
-OO
Dansyl-alanyl EP ε-N-Dansyl-α-N-tBoc-lysyl EP
Dansyl-glycyl EP Dansyl-methionyl EP
DNSExcitation at 330 nmEmission at 550 nm
5252
Fluorescent aminoacylation of tRNA
λexc 437 nm (α–Boc-ε-DNS-lysyl ethyl phosphate)λmax 500nm (free BocDNSLysEP)
One eq clearly distinct and sufficient
Reagent absorbance
5353
DNS Antibody
Specific for DNS Enhances fluorescence on binding
Blue shift when bound (from ~ 520 to ~ 450 nm)
Max fluorescence enhancement for DNS is about ten x
Steric hindrance - less enhancement
5454
Antibody detection of tRNA modification
+ Ab
‐ Ab
tRNA rxn + Anti-DNS Ab
Dans-Phe-tRNA forms ester
o-tRNA rxn + Anti-DNS Ab
N
N N
N
O
NH
ribose
H3C
OCH3
O
H3CO
O
Y
In Phe-tRNA
5555
Val-tRNA antibody detection
+ DNS‐Ab
No Ab
5656
o-tRNA with DNS-Val-EP
No diol – no reaction – retains DNS fluorescence of reactants only
+ Ab
- Ab
57
Tested aminoacyl phosphates
O2NHN
O
O
O
O
PO-
OO
HNO
OP
O
O
O-
O
OHO
HNO
O
O
O
PO-
OO
FHN
O
O
O
O
PO-
OO
Boc-tyrosyl ethyl phosphate (BocTEP)
Boc-4-fluorophenylalanyl ethyl phosphate(BocFPEP)
Boc-4-nitrophenylalanyl ethyl phosphate
Boc-phenylalanyl ethyl phosphate
NHO
O
N
SO OPO-
OO
N
SO ONH
NHBocO
O
P-O OO
N
SO O
NHO
OP
O
O
O-
DNS-glycyl ethyl phosphate(DNSGlyEP)
Boc-DNS-lysyl ethyl phosphate(BocDNSLysEP)
DNS-phenylalanyl ethyl phosphate(DNSPheEP)
5858
Answers: Direct aminoacylation of tRNA Activate amino acids - Aminoacyl phosphate monoesters React with OH not NH – Lanthanide complexation (Lewis acid) Challenges
Selecting for 3’-terminal hydroxyls (~75 others!) – diol chelation, Mg+2 to block internal OH
Detection of product – F NMR and fluorescence Phosphate by-product complexes La+3 – add Mg+2
O P
OOEt
O-La+3
Incorporation into a protein Preparation of Translationally Competent tRNA by Direct
Chemical Acylation, Duffy and Dougherty, Org. Lett., 2010, 123776–3779
59
“If direct aminoacylation of full-length tRNA can produce translationally competent tRNAs, the synthesis of dCA, one of the most laborious aspects of the chemical aminoacylation strategy, would become unnecessary.”
Production detection – MALDI
60
MALDI mass spectra of THG73 tRNA before (top) and after (below) exposure to La3+-mediated acylation conditions using leucine derivative 1.
“Applying the reported La3+-mediated acylation conditions to uridine and cytidine with activated amino acids such as 1−4gave 2′/3′ esters in low overall conversion (5−10% as measured by LC/MS). Increasing the equivalents of the aaEPs and La3+ to a large excess (≤20 equiv) with respect to the nucleoside improved the conversion (25−50%, data not shown). Using large excesses of aaEPs introduced solubility issues that could be alleviated by use of DMSO as a cosolvent.”
Aminoacylation without protecting group
61
Amino group is “protected” by protonation
Sohyoung Her
S. Her, R. Kluger, Org. Biomol. Chem. 2011 9, 676
The reaction of uridine with PheEP in the presence of lanthanum triflate is complete in less than one minute
Identification of esters Esters resist hydrolysis Yields > 60% Scramble positions Spectra after 30 minutes are shown for initially pure samples
62
1H NMR of two phenylalanine monoesters of uridine separated by reversed phase HPLC (A: Ester 1, B: Ester 2) Note that the 2’ and 3’-esters equilibrate
1’-proton
2’ ester, C1’-Hpyrimidine
3’ ester, C1’-H
Complementary aminoacylation methods Biomimetic
Side chains (and ester) are potentially unlimited
Catalytic La+3, Mg+2
Aminoacyl phosphate esters are readily prepared and stable
Enzymatic Highly efficient and
catalytic Side chain tolerance
depends on evolved specificity
ATP activation
63
Extensions in progress
tRNA reactions and detection Efficient catalyst utilization Chelation-controlled monoacylation of
carbohydrates
64
6565
Thanks
Richard Loo Vince Mazza Lisa Cameron Svetlana Tzvetkova Sohyoung Her