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Towards the Concise Syntheses of Selenium- and Tellurium-Containing Tryptophan Analogs for the Elucidation of Protein

Structure and Function Ryan Agh§¶, Ambrose Rice§¶, Duane M. Hatch§¶, Ricardo Martí-Arbona¶, L.A. “Pete” Silks¶

¶Bioscience Division, Group B11, Los Alamos National Laboratory, Los Alamos, NM, 87545 USA §Department of Chemistry & Physics, Belmont University, Nashville, TN, 37212

Bioscience

Innovation in

Health and Security

1. Welch, M.; Philips, R.S., Heterocyl. Com., 1999, 5, 305-310.

2. Hatch, D. M. et al. Current Org. Chem. 2004. 47-64.

3. Hatch, Duane. Tenn. Tech. Univ. 2003. pp. 4-8, 19-70.

4. Boles, J.O.; Henderson, J.; Hatch, D.; Silks, L.A. Biochem.

Biophys. Res. Comm. 2002. 257-261

References

Conclusions

• We have successfully validated our proposed synthesis

• We have analyzed all intermediates and products via 1H, 13C, 77Se, and 125Te NMR spectroscopy.

• We have successfully bio-incorporated SeTrp into a model

protein.4

Acknowledgements • Scott Robbins (LANL)

• Kirill Balatsky (LANL)

• Katrina Barnett (Belmont University)

• Belmont University Department of Chemistry and Physics

• Bioscience Division, B-11, LANL

• Partial funding from DOE Visiting Faculty Program 2015

• LANL Student Programs Office

Benefits

• Increase the number of available unnatural amino acids for

protein structure/function elucidation.

• Tryptophan is often found near or leading into the active

site of a protein, thereby allowing for interrogation of the

environment around that residue by NMR spectroscopy.

• Se-Trp and Te-Trp are less fluxional than Se-Met giving

rise to increased stability.

Future Work

• Continue scaling up the synthesis

and refining methods.

• Characterize catalytic activity of

Lignin Peroxidase with Trp

analogs.

• Improve stability of intermediate

compounds

• Get crystallographic data and

resolve the 3D structure of

selected proteins.

• Investigate catalytic activities of

SeTrp and TeTrp containing

proteins.

Unclassified 'LA-UR-15-25780'

Background

Since the 1990’s, Se-Methionine (Se-Met) has been invaluable in

the elucidation of 3D-protein structures. There have also been

published results of a successful synthesis of Se-Trp (Scheme

1).1 However, this synthetic route is an expensive one, with low

Scheme 1: Phillip’s Modified

Paulmier Synthesis1

yield, and the

inability to label the

compound with the 77Se isotope. Our

synthetic strategy is

more refined and

allows for 77Se

insertion. (Scheme

2).2,3

1. Determine the viability of using

this route for large production of

6H-selenolo[2,3-b]pyrrole.

2. Synthesize 6H-telluro[2,3-

b]pyrrole utilizing this method.

3. Maximize the enzymatic coupling

of the serine side chain using

purified Tryptophan Synthase.

X = Se or Te

Scheme 2: LANL Synthesis2,3

Goals

Trp

Synthase

Figure 1: L-Tryptophan

Figure 2: [6,7]SeTrp or [6,7]TeTrp

Overview

We worked on an improved synthesis which forms an amino

acid (L-Tryptophan, Figure 1) analog. Our synthesis can be

used to insert either Selenium or Tellurium into the amino acid

(Figure 2). This was done to investigate protein structure and

catalytic activity via X-Ray Crystallography (Figure 3) and

NMR Spectroscopy.

89.00%

10.00% 0.70%

0.30%

X-Ray

Crystallography

NMR Spectroscopy

Electron

Microscopy

Other Methods

Figure 3: Methods for Solving Protein Structure X = Se or Te

No Hh, Hi, or Hj

Syntheses

6H-selenolo[2,3-b]pyrrole 6H-telluro[2,3-b]pyrrole

63% Yield

82% Yield 75%Yield

80% Yield

80% Yield

He

Hg

Hd Hc

Hb

Ha

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

f1 (ppm)

1 Hj

Hh

Hi

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

f1 (ppm)

1

Hj

Hh

Hi

Hf

He

Hg

Hf

77Se-NMR Chemical Shifts

No He, Hf, or Hg

169.97 ppm 507.83 ppm 437.44 ppm

No Hh, Hi, or Hj